Combination therapy with an antitumor alkaloid

ABSTRACT

The present invention relates to the combination of PM01183 with several anticancer drugs, in particular other anticancer drugs selected from antitumor platinum coordination complexes, antimetabolites, mitotic inhibitors, anticancer antibiotics, topoisomerase I and/or II inhibitors, proteasome inhibitors, histone deacetylase inhibitors, nitrogen mustard alkylating agents, nitrosourea alkylating agents, nonclassical alkylating agents, estrogen antagonists, androgen antagonists, mTOR inhibitors, tyrosine kinase inhibitors, and other agents selected from aplidine, ET-743, PM02734 and PM00104, and the use of these combinations in the treatment of cancer.

FIELD OF THE INVENTION

The present invention relates to the combination of PM01183 with otheranticancer drugs, in particular other anticancer drugs selected fromantitumor platinum coordination complexes, antimetabolites, mitoticinhibitors, anticancer antibiotics, topoisomerase I and/or IIinhibitors, proteasome inhibitors, histone deacetylase inhibitors,nitrogen mustard alkylating agents, nitrosourea alkylating agents,nonclassical alkylating agents, estrogen antagonists, androgenantagonists, mTOR inhibitors, tyrosine kinase inhibitors, and otheragents selected from aplidine, ET-743, PM02734, and PM00104 and the useof these combinations in the treatment of cancer.

BACKGROUND OF THE INVENTION

Cancer develops when cells in a part of the body begin to grow out ofcontrol. Although there are many kinds of cancer, they all arise fromout-of-control growth of abnormal cells. Cancer cells can invade nearbytissues and can spread through the bloodstream and lymphatic system toother parts of the body. There are several main types of cancer.Carcinoma is a malignant neoplasm, which is an uncontrolled andprogressive abnormal growth, arising from epithelial cells. Epithelialcells cover internal and external surfaces of the body, includingorgans, lining of vessels, and other small cavities. Sarcoma is cancerarising from cells in bone, cartilage, fat, muscle, blood vessels, orother connective or supportive tissue. Leukemia is cancer that arises inblood-forming tissue such as the bone marrow, and causes large numbersof abnormal blood cells to be produced and enter the bloodstream.Lymphoma and multiple myeloma are cancers that arise from cells of theimmune system.

In addition, cancer is invasive and tends to infiltrate the surroundingtissues and give rise to metastases. It can spread directly intosurrounding tissues and also may be spread through the lymphatic andcirculatory systems to other parts of the body.

Many treatments are available for cancer, including surgery andradiation for localised disease, and chemotherapy. However, the efficacyof available treatments for many cancer types is limited, and new,improved forms of treatment showing clinical benefits are needed. Thisis especially true for those patients presenting with advanced and/ormetastatic disease and for patients relapsing with progressive diseaseafter having been previously treated with established therapies whichbecome ineffective or intolerable due to acquisition of resistance or tolimitations in administration of the therapies due to associatedtoxicities.

Since the 1950s, significant advances have been made in thechemotherapeutic management of cancer. Unfortunately, more than 50% ofall cancer patients either do not respond to initial therapy orexperience relapse after an initial response to treatment and ultimatelydie from progressive metastatic disease. Thus, the ongoing commitment tothe design and discovery of new anticancer agents is criticallyimportant.

Chemotherapy, in its classic form, has been focused primarily on killingrapidly proliferating cancer cells by targeting general cellularmetabolic processes, including DNA, RNA, and protein biosynthesis.Chemotherapy drugs are divided into several groups based on how theyaffect specific chemical substances within cancer cells, which cellularactivities or processes the drug interferes with, and which specificphases of the cell cycle the drug affects. The most commonly used typesof chemotherapy drugs include: DNA-alkylating drugs (such ascyclophosphamide, ifosfamide, cisplatin, carboplatin, dacarbazine),antimetabolites (5-fluorouracil, capecitabine, 6-mercaptopurine,methotrexate, gemcitabine, cytarabine, fludarabine), mitotic inhibitors(such as paclitaxel, docetaxel, vinblastine, vincristine), anticancerantibiotics (such as daunorubicin, doxorubicin, epirubicin, idarubicin,mitoxantrone), topoisomerase I and/or II inhibitors (such as topotecan,irinotecan, etoposide, teniposide), and hormone therapy (such astamoxifen, flutamide).

The ideal antitumor drug would kill cancer cells selectively, with awide index relative to its toxicity towards non-cancer cells and itwould also retain its efficacy against cancer cells, even afterprolonged exposure to the drug. Unfortunately, none of the currentchemotherapies with these agents posses an ideal profile. Most possesvery narrow therapeutic indexes and, in addition, cancerous cellsexposed to slightly sublethal concentrations of a chemotherapeutic agentmay develop resistance to such an agent, and quite oftencross-resistance to several other antitumor agents.

PM01183, also known as tryptamicidin, is a synthetic alkaloid which iscurrently in clinical trials for the treatment of cancer, and has thefollowing chemical structure:

PM01183 has demonstrated a highly potent in vitro activity against solidand non-solid tumour cell lines as well as a significant in vivoactivity in several xenografted human tumor cell lines in mice, such asthose for breast, kidney and ovarian cancer. PM01183 exerts itsanticancer effects through the covalent modification of guanines in theDNA minor groove that eventually give rise to DNA double-strand break,S-phase arrest and apoptosis in cancer cells. Further informationregarding this compound can be found in WO 03/01427; 100^(th) AACRAnnual Meeting, Apr. 18-22, 2009, Denver, Colo., Abstract Nr. 2679 andAbstract Nr. 4525; and Leal J F M et al. Br. J. Pharmacol. 2010, 161,1099-1110.

Since cancer is a leading cause of death in animals and humans, severalefforts have been and are still being undertaken in order to obtain atherapy active and safe to be administered to patients suffering from acancer. The problem to be solved by the present invention is to provideanticancer therapies that are useful in the treatment of cancer.

SUMMARY OF THE INVENTION

The present invention establishes that PM01183 potentiates the antitumoractivity of other anticancer agents, in particular other anticancerdrugs selected from antitumor platinum coordination complexes,antimetabolites, mitotic inhibitors, anticancer antibiotics,topoisomerase I and/or II inhibitors, proteasome inhibitors, histonedeacetylase inhibitors, nitrogen mustard alkylating agents, nitrosoureaalkylating agents, nonclassical alkylating agents, estrogen antagonists,androgen antagonists, mTOR inhibitors, tyrosine kinase inhibitors, andother agents selected from aplidine, ET-743, PM02734 and PM00104.Therefore PM01183 and said other anticancer agents can be successfullyused in combination therapy for the treatment of cancer.

Thus, this invention is directed to pharmaceutical compositions, kits,methods for the treatment of cancer using these combination therapiesand uses of both drugs in the treatment of cancer and in the manufactureof medicaments for combination therapies.

In accordance with one aspect of this invention, we provide effectivecombination therapies for the treatment of cancer based on PM01183, or apharmaceutically acceptable salt thereof, and using another anticancerdrug as defined above.

In another embodiment, the invention is directed to PM01183, or apharmaceutically acceptable salt thereof, for use in the treatment ofcancer comprising administering a therapeutically effective amount ofPM01183, or a pharmaceutical acceptable salt thereof, in combinationwith a therapeutically effective amount of another anticancer drug.

In another embodiment, the invention encompasses a method of treatingcancer comprising administering to a patient in need of such treatment atherapeutically effective amount of PM01183, or a pharmaceuticallyacceptable salt thereof, and a therapeutically effective amount ofanother anticancer drug.

In another aspect, the invention encompasses a method of increasing orpotentiating the therapeutic efficacy of an anticancer drug in thetreatment of cancer, which comprises administering to a patient in needthereof a therapeutically effective amount of PM01183, or apharmaceutically acceptable salt thereof, in conjunction with this otheranticancer drug.

In another embodiment, the invention encompasses the use of PM01183, ora pharmaceutically acceptable salt thereof, for the manufacture of amedicament for the treatment of cancer by combination therapy employingPM01183, or a pharmaceutically acceptable salt thereof, with anotheranticancer drug.

In a further aspect, the invention encompasses a pharmaceuticalcomposition comprising PM01183, or a pharmaceutically acceptable saltthereof, and/or another anticancer drug, and a pharmaceuticallyacceptable carrier, to be used in combination therapy for the treatmentof cancer.

The invention also encompasses a kit for use in the treatment of cancerwhich comprises a dosage form of PM01183, or a pharmaceuticallyacceptable salt thereof, and/or a dosage form of another anticancerdrug, and instructions for the use of both drugs in combination.

In one preferred aspect, the present invention is concerned withsynergistic combinations of PM01183, or a pharmaceutically acceptablesalt thereof, with another anticancer drug.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1-20. In vitro activity data of PM01183 in combination withoxaliplatin, 5-fluorouracil, gemcitabine, paclitaxel, docetaxel,vincristine, daunorubicin, mitomycin C, actinomycin D, topotecan,etoposide, bortezomib, vorinostat, cyclophosphamide, carmustine,dacarbazine, temsirolimus, erlotinib, ET-743 and PM00104 respectivelyagainst A549 cells.

FIG. 21-41. In vitro activity data of PM01183 in combination withcisplatin, oxaliplatin, cytarabine, gemcitabine, docetaxel, vincristine,vinorelbine, daunorubicin, mitomycin C, actinomycin D, topotecan,etoposide, vorinostat, cyclophosphamide, dacarbazine, temsirolimus,erlotinib, aplidine, ET-743, PM02734 and PM00104 respectively againstA673 cells.

FIG. 42-56. In vitro activity data of PM01183 in combination withcisplatin, 5-fluorouracil, cytarabine, methotrexate, daunorubicin,doxorubicin, mitomycin C, topotecan, irinotecan, etoposide, dacarbazine,temsirolimus, ET-743, PM02734 and PM00104 respectively against SK-MEL-2cells.

FIG. 57-80. In vitro activity data of PM01183 in combination withcisplatin, oxaliplatin, 5-fluorouracil, cytarabine, gemcitabine,methotrexate, docetaxel, paclitaxel, vinorelbine, daunorubicin,doxorubicin, mitomycin C, actinomycin D, topotecan, irinotecan,etoposide, bortezomib, vorinostat, flutamide, temsirolimus, erlotinib,ET-743, PM02734 and PM00104 respectively against PC-3 cells.

FIG. 81-98. In vitro activity data of PM01183 in combination withcisplatin, oxaliplatin, cytarabine, gemcitabine, methotrexate,daunorubicin, doxorubicin, actinomycin D, topotecan, irinotecan,etoposide, bortezomib, vorinostat, temsirolimus, erlotinib, ET-743,PM02734 and PM00104 respectively against PANC-1 cells.

FIG. 99-123. In vitro activity data of PM01183 in combination withcisplatin, oxaliplatin, 5-fluorouracil, cytarabine, gemcitabine,methotrexate, paclitaxel, vincristine, vinorelbine, daunorubicin,doxorubicin, actinomycin D, topotecan, irinotecan, etoposide,bortezomib, vorinostat, cyclophosphamide, dacarbazine, temsirolimus,erlotinib, aplidine, ET-743, PM02734 and PM00104 respectively againstHGC-27 cells.

FIG. 124-150. In vitro activity data of PM01183 in combination withcisplatin, oxaliplatin, 5-fluorouracil, cytarabine, gemcitabine,methotrexate, docetaxel, paclitaxel, vincristine, vinorelbine,daunorubicin, doxorubicin, actinomycin D, mitomycin C, topotecan,irinotecan, etoposide, vorinostat, cyclophosphamide, carmustine,dacarbazine, temsirolimus, erlotinib, aplidine, ET-743, PM02734 andPM00104 respectively against IGROV-1 cells.

FIG. 151-170. In vitro activity data of PM01183 in combination withcisplatin, oxaliplatin, 5-fluorouracil, cytarabine, gemcitabine,methotrexate, docetaxel, paclitaxel, vincristine, vinorelbine,daunorubicin, doxorubicin, topotecan, irinotecan, etoposide, bortezomib,cyclophosphamide, erlotinib, ET-743 and PM00104 respectively againstHEP-G2 cells.

FIG. 171-197. In vitro activity data of PM01183 in combination withcisplatin, oxaliplatin, 5-fluorouracil, cytarabine, gemcitabine,methotrexate, docetaxel, paclitaxel, vincristine, vinorelbine,daunorubicin, doxorubicin, actinomycin D, mitomycin C, topotecan,irinotecan, etoposide, vorinostat, cyclophosphamide, carmustine,dacarbazine, tamoxifen, temsirolimus, erlotinib, ET-743, PM02734 andPM00104 respectively against MDA-MB-231 cells.

FIG. 198-219. In vitro activity data of PM01183 in combination withcisplatin, oxaliplatin, 5-fluorouracil, cytarabine, gemcitabine,docetaxel, vinorelbine, daunorubicin, doxorubicin, actinomycin D,mitomycin C, topotecan, irinotecan, etoposide, bortezomib, vorinostat,cyclophosphamide, dacarbazine, temsirolimus, erlotinib, aplidine andPM02734 respectively against HT-29 cells.

FIG. 220-242. In vitro activity data of PM01183 in combination withcisplatin, 5-fluorouracil, cytarabine, gemcitabine, methotrexate,docetaxel, vincristine, vinorelbine, daunorubicin, doxorubicin,actinomycin D, mitomycin C, topotecan, irinotecan, etoposide,vorinostat, cyclophosphamide, dacarbazine, erlotinib, aplidine, ET-743,PM02734 and PM00104 respectively against RXF-393 cells.

FIG. 243-262. In vitro activity data of PM01183 in combination withcisplatin, oxaliplatin, 5-fluorouracil, gemcitabine, methotrexate,docetaxel, vincristine, daunorubicin, doxorubicin, topotecan,irinotecan, etoposide, bortezomib, vorinostat, dacarbazine,temsirolimus, erlotinib, aplidine, ET-743 and PM02734 respectivelyagainst U87-MG cells.

FIG. 263. Tumor volume evaluation of A2780 tumors in mice treated withplacebo, PM01183, paclitaxel and PM01183 plus paclitaxel.

FIG. 264. Tumor volume evaluation of A2780 tumors in mice treated withplacebo, PM01183, vinorelbine and PM01183 plus vinorelbine.

FIG. 265. Tumor volume evaluation of A2780 tumors in mice treated withplacebo, PM01183, doxorubicin and PM01183 plus doxorubicin.

FIG. 266. Tumor volume evaluation of HGC-27 tumors in mice treated withplacebo, PM01183, cisplatin and PM01183 plus cisplatin.

FIG. 267. Tumor volume evaluation of HGC-27 tumors in mice treated withplacebo, PM01183, 5-fluorouracil and PM01183 plus 5-fluorouracil.

FIG. 268. Tumor volume evaluation of SW1990 tumors in mice treated withplacebo, PM01183, gemcitabine and PM01183 plus gemcitabine.

FIG. 269. Tumor volume evaluation of U87-MG tumors in mice treated withplacebo, PM01183, temozolomide and PM01183 plus temozolomide.

FIG. 270. Tumor volume evaluation of H460 tumors in mice treated withplacebo, PM01183, irinotecan and PM01183 plus irinotecan.

FIG. 271. Tumor volume evaluation of HT1080 tumors in mice treated withplacebo, PM01183, dacarbazine and PM01183 plus dacarbazine.

FIG. 272. Tumor volume evaluation of HT-29 tumors in mice treated withplacebo, PM01183, irinotecan and PM01183 plus irinotecan.

FIG. 273. Effects of the combination of PM01183 with methotrexate inJURKAT cell line.

FIG. 274. Effects of the combination of PM01183 with methotrexate inMOLT-4 cell line.

FIG. 275. Effects of the combination of PM01183 with daunorubicin inJURKAT cell line.

FIG. 276. Effects of the combination of PM01183 with aplidine in JURKATcell line.

FIG. 277. Effects of the combination of PM01183 with aplidine in MOLT-4cell line.

FIG. 278. Effects of the combination of PM01183 with ET-743 in JURKATcell line.

FIG. 279. Effects of the combination of PM01183 with ET-743 in MOLT-4cell line.

FIG. 280. Effects of the combination of PM01183 with PM00104 in JURKATcell line.

FIG. 281. Effects of the combination of PM01183 with PM00104 in MOLT-4cell line.

FIG. 282. Effects of the combination of PM01183 with PM02734 in JURKATcell line.

FIG. 283. Effects of the combination of PM01183 with PM02734 in MOLT-4cell line.

FIG. 284. Effects of the combination of PM01183 with cytarabine in RAMOScell line.

FIG. 285. Effects of the combination of PM01183 with methotrexate inRAMOS cell line.

FIG. 286. Effects of the combination of PM01183 with methotrexate inU-937 cell line.

FIG. 287. Effects of the combination of PM01183 with gemcitabine inRAMOS cell line.

FIG. 288. Effects of the combination of PM01183 with gemcitabine inU-937 cell line.

FIG. 289. Effects of the combination of PM01183 with daunorubicin inRAMOS cell line.

FIG. 290. Effects of the combination of PM01183 with daunorubicin inU-937 cell line.

FIG. 291. Effects of the combination of PM01183 with ET-743 in RAMOScell line.

FIG. 292. Effects of the combination of PM01183 with ET-743 in U-937cell line.

FIG. 293. Effects of the combination of PM01183 with PM00104 in RAMOScell line.

FIG. 294. Effects of the combination of PM01183 with PM00104 in U-937cell line.

FIG. 295. Effects of the combination of PM01183 with PM02734 in RAMOScell line.

FIG. 296. Effects of the combination of PM01183 with PM02734 in U-937cell line.

DETAILED DESCRIPTION OF THE INVENTION

We surprisingly found that PM01183 greatly enhances the anticanceractivity of other anticancer drugs when these anticancer drugs arecombined with PM01183. Thus, the present invention is directed toprovide an efficacious treatment of cancer based on the combination ofPM01183, or a pharmaceutically acceptable salt thereof, with anotheranticancer drug.

In the present application, by “cancer” it is meant to include tumors,neoplasias, and any other malignant disease having as cause malignanttissue or cells.

The term “treating”, as used herein, unless otherwise indicated, meansreversing, alleviating, or inhibiting the progress of the disease orcondition to which such term applies, or one or more symptoms of suchdisorder or condition. The term “treatment”, as used herein, unlessotherwise indicated, refers to the act of treating as “treating” isdefined immediately above.

The term “combination” as used throughout the specification, is meant toencompass the administration to a patient suffering from cancer of thereferred therapeutic agents in the same or separate pharmaceuticalformulations, and at the same time or at different times. If thetherapeutic agents are administered at different times they should beadministered sufficiently close in time to provide for the potentiatingor synergistic response to occur.

As mentioned above, PM01183 is a synthetic alkaloid, having thefollowing structure:

The term “PM01183” is intended here to cover any pharmaceuticallyacceptable salt, solvate, hydrate, prodrug, or any other compound which,upon administration to the patient is capable of providing (directly orindirectly) the compound as described herein. The preparation of salts,solvates, hydrates, and prodrugs can be carried out by methods known inthe art.

Pharmaceutically acceptable salts can be synthesized from the parentcompound, which contains a basic or acidic moiety, by conventionalchemical methods. Generally, such salts are, for example, prepared byreacting the free acid or base forms of these compounds with astoichiometric amount of the appropriate base or acid in water or in anorganic solvent or in a mixture of the two. Generally, nonaqueous medialike ether, ethyl acetate, ethanol, isopropanol or acetonitrile arepreferred. Examples of the acid addition salts include mineral acidaddition salts such as, for example, hydrochloride, hydrobromide,hydroiodide, sulphate, nitrate, phosphate, and organic acid additionsalts such as, for example, acetate, trifluoroacetate, maleate,fumarate, citrate, oxalate, succinate, tartrate, malate, mandelate,methanesulphonate and p-toluenesulphonate. Examples of the alkaliaddition salts include inorganic salts such as, for example, sodium,potassium, calcium and ammonium salts, and organic alkali salts such as,for example, ethylenediamine, ethanolamine, N,N-dialkylenethanolamine,triethanolamine and basic aminoacids salts.

Any compound that is a prodrug of PM01183 is within the scope and spiritof the invention. The term “prodrug” is used in its broadest sense andencompasses those derivatives that are converted in vivo to PM01183. Theprodrug can hydrolyze, oxidize, or otherwise react under biologicalconditions to provide PM01183. Examples of prodrugs include, but are notlimited to, derivatives and metabolites of PM01183 that includebiohydrolyzable moeities such as biohydrolyzable amides, biohydrolyzableesters, biohydrolyzable carbamates, biohydrolyzable carbonates,biohydrolyzable ureides, and biohydrolyzable phosphate analogues.Prodrugs can typically be prepared using well-known methods, such asthose described by Burger in “Medicinal Chemistry and Drug Discovery”6th ed. (Donald J. Abraham ed., 2001, Wiley) and “Design andApplications of Prodrugs” (H. Bundgaard ed., 1985, Harwood AcademicPublishers).

In addition, any drug referred to herein may be in amorphous form orcrystalline form either as free compound or as solvates (e.g. hydrates)and it is intended that both forms are within the scope of the presentinvention. Methods of solvation are generally known within the art.

Moreover, PM01183 for use in accordance with the present invention maybe prepared following the synthetic process such as the one disclosed inWO 03/014127, which is incorporated herein by reference.

Pharmaceutical compositions of PM01183, or of a pharmaceuticallyacceptable salt thereof, that can be used include solutions,suspensions, emulsions, lyophilised compositions, etc., with suitableexcipients for intravenous administration. Preferably, PM01183 may besupplied and stored as a sterile lyophilized product, comprising PM01183and excipients in a formulation adequate for therapeutic use. Forfurther guidance on pharmaceutical compositions of PM01183, or apharmaceutically acceptable salt thereof, see for example theformulations described in WO 2006/046079, which is incorporated hereinby reference.

Administration of PM01183, or a pharmaceutically acceptable saltthereof, or pharmaceutical compositions comprising the compound ispreferably by intravenous infusion. Infusion times of up to 72 hours canbe used, more preferably between 1 and 24 hours, with either about 1hour or about 3 hours most preferred. Short infusion times which allowtreatment to be carried out without an overnight stay in hospital areespecially desirable. However, infusion may be around 24 hours or evenlonger if required.

Preferably the administration of PM01183 is performed in cycles. In apreferred administration schedule an intravenous infusion of PM01183 isgiven to the patients the first week of each cycle and the patients areallowed to recover for the remainder of the cycle. The preferredduration of each cycle is of either 3 or 4 weeks. Multiple cycles can begiven as needed. Administration of PM01183, or a pharmaceuticallyacceptable salt thereof, by intravenous infusion during about 1 houronce every 3 weeks is the most preferred administration schedule,although other protocols can be devised as variations.

In the present invention, particularly preferred is the combination ofPM01183, or a pharmaceutically acceptable salt thereof, with anotheranticancer drug selected from antitumor platinum coordination complexes,antimetabolites, mitotic inhibitors, anticancer antibiotics,topoisomerase I and/or II inhibitors, proteasome inhibitors, histonedeacetylase inhibitors, nitrogen mustard alkylating agents, nitrosoureaalkylating agents, nonclassical alkylating agents, estrogen antagonists,androgen antagonists, mTOR inhibitors, tyrosine kinase inhibitors, andother agents selected from aplidine, ET-743, PM02734 and PM00104 in thetreatment of cancer.

Particularly preferred cancer types are those selected from lung cancer,sarcoma, malignant melanoma, bladder carcinoma, prostate cancer,pancreas carcinoma, thyroid cancer, gastric carcinoma, ovarian cancer,hepatoma (also known as liver cancer), breast cancer, colorectal cancer,kidney cancer, esophageal cancer, neuroblastoma, brain cancer, cervicalcancer, anal cancer, testicular cancer, leukemia, multiple myeloma andlymphoma.

In a preferred embodiment, the invention is directed to the combinationof PM01183, or a pharmaceutically acceptable salt thereof, with anantitumor platinum coordination complex in the treatment of cancer, andmore particularly in the treatment of a cancer selected from lungcancer, sarcoma, malignant melanoma, prostate cancer, pancreascarcinoma, gastric carcinoma, ovarian cancer, hepatoma, breast cancer,colorectal cancer, kidney cancer, brain cancer and lymphoma. Thischemotherapeutic group includes, but is not limited to cisplatin,oxaliplatin, carboplatin, triplatin tetranitrate (BBR3464), satraplatin,tetraplatin, ormiplatin, iproplatin, nedaplatin and lobaplatin.Particularly preferred is the combination of PM01183, or apharmaceutically acceptable salt thereof, with cisplatin, oxaliplatin,carboplatin, triplatin tetranitrate, satraplatin, tetraplatin,ormiplatin, iproplatin, nedaplatin and lobaplatin, and even morepreferred is the combination with cisplatin and oxaliplatin in thetreatment of cancer, and more particularly in the treatment of a cancerselected from lung cancer, sarcoma, malignant melanoma, prostate cancer,pancreas carcinoma, gastric carcinoma, ovarian cancer, hepatoma, breastcancer, colorectal cancer, kidney cancer and brain cancer.

In another preferred embodiment, the invention is directed to thecombination of PM01183, or a pharmaceutically acceptable salt thereof,with an antimetabolite in the treatment of cancer, and more particularlyin the treatment of a cancer selected from lung cancer, sarcoma,malignant melanoma, bladder carcinoma, prostate cancer, pancreascarcinoma, gastric carcinoma, ovarian cancer, hepatoma, breast cancer,colorectal cancer, kidney cancer, esophageal cancer, brain cancer, analcancer, leukaemia and lymphoma. This chemotherapeutic group includes,but is not limited to 5-fluorouracil, gemcitabine, cytarabine,capecitabine, decitabine, floxuridine, fludarabine, aminopterin,methotrexate, pemetrexed, raltitrexed, cladribine, clofarabine,mercaptopurine, pentostatin, and thioguanine. Particularly preferred isthe combination of PM01183, or a pharmaceutically acceptable saltthereof, with 5-fluorouracil, gemcitabine, cytarabine, capecitabine,decitabine, floxuridine, fludarabine, aminopterin, methotrexate,pemetrexed, raltitrexed, cladribine, clofarabine, mercaptopurine,pentostatin, and thioguanine, and even more preferred is the combinationwith 5-fluorouracil, gemcitabine, cytarabine and methotrexate in thetreatment of cancer, and more particularly in the treatment of a cancerselected from lung cancer, sarcoma, malignant melanoma, prostate cancer,pancreas carcinoma, gastric carcinoma, ovarian cancer, hepatoma, breastcancer, colorectal cancer, kidney cancer, brain cancer, leukemia andlymphoma.

In another preferred embodiment, the invention is directed to thecombination of PM01183, or a pharmaceutically acceptable salt thereof,with a mitotic inhibitor in the treatment of cancer, and moreparticularly in the treatment of a cancer selected from lung cancer,sarcoma, prostate cancer, gastric carcinoma, ovarian cancer, hepatoma,breast cancer, colorectal cancer, kidney cancer, brain cancer, leukemia,and lymphoma. This chemotherapeutic group includes, but is not limitedto paclitaxel, docetaxel, vinblastine, vincristine, vindesine, andvinorelbine. Particularly preferred is the combination of PM01183, or apharmaceutically acceptable salt thereof, with paclitaxel, docetaxel,vinblastine, vincristine, vindesine, and vinorelbine, and even morepreferred is the combination with paclitaxel, docetaxel, vincristine andvinorelbine in the treatment of cancer, and more particularly in thetreatment of a cancer selected from lung cancer, sarcoma, prostatecancer, gastric carcinoma, ovarian cancer, hepatoma, breast cancer,colorectal cancer, kidney cancer and brain cancer.

In another preferred embodiment, the invention is directed to thecombination of PM01183, or a pharmaceutically acceptable salt thereof,with an anticancer antibiotic in the treatment of cancer, and moreparticularly in the treatment of lung cancer, sarcoma, malignantmelanoma, bladder carcinoma, prostate cancer, pancreas carcinoma,thyroid cancer, gastric carcinoma, ovarian cancer, hepatoma, breastcancer, colorectal cancer, kidney cancer, neuroblastoma, brain cancer,anal cancer, testicular cancer, leukemia, multiple myeloma and lymphoma.This chemotherapeutic group includes, but is not limited todaunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone,pixantrone, valrubicin, mitomycin C, bleomycin, actinomycin A andmithramycin. Particularly preferred is the combination of PM01183, or apharmaceutically acceptable salt thereof, with daunorubicin,doxorubicin, epirubicin, idarubicin, mitoxantrone, pixantrone,valrubicin, mitomycin C, bleomycin, actinomycin D and mithramycin, andeven more preferred is the combination with daunorubicin, doxorubicin,mitomycin C and actinomycin D in the treatment of cancer, and moreparticularly in the treatment of lung cancer, sarcoma, malignantmelanoma, prostate cancer, pancreas carcinoma, gastric carcinoma,ovarian cancer, hepatoma, breast cancer, colorectal cancer, kidneycancer, brain cancer, leukemia and lymphoma.

In another preferred embodiment, the invention is directed to thecombination of PM01183, or a pharmaceutically acceptable salt thereof,with a topoisomerase I and/or II inhibitor in the treatment of cancer,and more particularly in the treatment of lung cancer, sarcoma,malignant melanoma, prostate cancer, pancreas carcinoma, gastriccarcinoma, ovarian cancer, hepatoma, breast cancer, colorectal cancer,kidney cancer, neuroblastoma, brain cancer, cervical cancer, testicularcancer, leukemia and lymphoma. This chemotherapeutic group includes, butis not limited to topotecan, SN-38, irinotecan, camptothecin, rubitecan,etoposide, amsacrine and teniposide. Particularly preferred is thecombination of PM00104, or a pharmaceutically acceptable salt thereof,with topotecan, SN-38, irinotecan, camptothecin, rubitecan, etoposide,amsacrine and teniposide, and even more preferred is the combinationwith topotecan, irinotecan and etoposide in the treatment of cancer, andmore particularly in the treatment of lung cancer, sarcoma, malignantmelanoma, prostate cancer, pancreas carcinoma, gastric carcinoma,ovarian cancer, hepatoma, breast cancer, colorectal cancer, kidneycancer, and brain cancer.

In another preferred embodiment, the invention is directed to thecombination of PM01183, or a pharmaceutically acceptable salt thereof,with a proteosome inhibitor in the treatment of cancer, and moreparticularly in the treatment of lung cancer, prostate cancer, pancreascarcinoma, gastric carcinoma, hepatoma, colorectal cancer, brain cancer,multiple myeloma and lymphoma. This chemotherapeutic group includes, butis not limited to bortezomib, disulfiram, epigallocatechin gallate, andsalinosporamide A. Particularly preferred is the combination of PM01183,or a pharmaceutically acceptable salt thereof, with bortezomib,disulfiram, epigallocatechin gallate, and salinosporamide A, and evenmore preferred is the combination with bortezomib in the treatment ofcancer, and more particularly in the treatment of lung cancer, prostatecancer, pancreas carcinoma, gastric carcinoma, hepatoma, colorectalcancer and brain cancer.

In another preferred embodiment, the invention is directed to thecombination of PM01183, or a pharmaceutically acceptable salt thereof,with a histone deacetylase inhibitor in the treatment of cancer, andmore particularly in the treatment of lung cancer, sarcoma, prostatecancer, pancreas carcinoma, gastric carcinoma, ovarian cancer, breastcancer, colorectal cancer, kidney cancer, brain cancer and lymphoma.This chemotherapeutic group includes, but is not limited to romidepsin,panobinostat, vorinostat, mocetinostat, belinostat, entinostat,resminostat, PCI-24781, AR-42, CUDC-101, and valproic acid. Particularlypreferred is the combination of PM01183, or a pharmaceuticallyacceptable salt thereof, with romidepsin, panobinostat, vorinostat,mocetinostat, belinostat, entinostat, resminostat, PCI-24781, AR-42,CUDC-101, and valproic acid, and even more preferred is the combinationwith vorinostat in the treatment of cancer, and more particularly in thetreatment of lung cancer, sarcoma, prostate cancer, pancreas carcinoma,gastric carcinoma, ovarian cancer, breast cancer, colorectal cancer,kidney cancer and brain cancer.

In another preferred embodiment, the invention is directed to thecombination of PM01183, or a pharmaceutically acceptable salt thereof,with a nitrogen mustard alkylating agent in the treatment of cancer, andmore particularly in the treatment of lung cancer, sarcoma, bladdercarcinoma, gastric carcinoma, ovarian cancer, hepatoma, breast cancer,colorectal cancer, kidney cancer, leukemia, multiple myeloma andlymphoma. This chemotherapeutic group includes, but is not limited tomelphalan, ifosfamide, chlorambucil, cyclophosphamide, mechlorethamine,uramustine, estramustine and bendamustine. Particularly preferred is thecombination of PM01183, or a pharmaceutically acceptable salt thereof,with melphalan, ifosfamide, chlorambucil, cyclophosphamide,mechlorethamine, uramustine, estramustine and bendamustine, and evenmore preferred is the combination with cyclophosphamide in the treatmentof cancer, and more particularly in the treatment of lung cancer,sarcoma, gastric carcinoma, ovarian cancer, hepatoma, breast cancer,colorectal cancer and kidney cancer.

In another preferred embodiment, the invention is directed to thecombination of PM01183, or a pharmaceutically acceptable salt thereof,with a nitrosourea alkylating agent in the treatment of cancer, and moreparticularly in the treatment of lung cancer, ovarian cancer, breastcancer, brain cancer, multiple myeloma and lymphoma. Thischemotherapeutic group includes, but is not limited to lomustine,semustine, carmustine, fotemustine and streptozotocin. Particularlypreferred is the combination of PM01183, or a pharmaceuticallyacceptable salt thereof, with lomustine, semustine, carmustine,fotemustine and streptozotocin, and even more preferred is thecombination with carmustine in the treatment of cancer, and moreparticularly in the treatment of lung cancer, ovarian cancer and breastcancer.

In another preferred embodiment, the invention is directed to thecombination of PM01183, or a pharmaceutically acceptable salt thereof,with a nonclassical alkylating agent in the treatment of cancer, andmore particularly in the treatment of lung cancer, sarcoma, malignantmelanoma, pancreas carcinoma, gastric carcinoma, ovarian cancer, breastcancer, colorectal cancer, kidney cancer, brain cancer, leukemia andlymphoma. This chemotherapeutic group includes, but is not limited toprocarbazine, dacarbazine, temozolomide and altretamine. Particularlypreferred is the combination of PM01183, or a pharmaceuticallyacceptable salt thereof, with procarbazine, dacarbazine, temozolomideand altretamine, and even more preferred is the combination withdacarbazine and tezolomide in the treatment of lung cancer, sarcoma,malignant melanoma, gastric carcinoma, ovarian cancer, breast cancer,colorectal cancer, kidney cancer and brain cancer.

In another preferred embodiment, the invention is directed to thecombination of PM01183, or a pharmaceutically acceptable salt thereof,with an estrogen antagonist in the treatment of cancer, and moreparticularly in the treatment of breast cancer. This chemotherapeuticgroup includes, but is not limited to toremifene, fulvestrant, tamoxifenand nafoxidine. Particularly preferred is the combination of PM01183, ora pharmaceutically acceptable salt thereof, with toremifene,fulvestrant, tamoxifen and nafoxidine, and even more preferred is thecombination with tamoxifen in the treatment of breast cancer.

In another preferred embodiment, the invention is directed to thecombination of PM01183, or a pharmaceutically acceptable salt thereof,with an androgen antagonist in the treatment of cancer, and moreparticularly in the treatment of prostate cancer. This chemotherapeuticgroup includes, but is not limited to bicalutamide, flutamide, MDV3100and nilutamide. Particularly preferred is the combination of PM01183, ora pharmaceutically acceptable salt thereof, with bicalutamide,flutamide, MDV3100 and nilutamide, and even more preferred is thecombination with flutamide in the treatment of prostate cancer.

In another preferred embodiment, the invention is directed to thecombination of PM01183, or a pharmaceutically acceptable salt thereof,with a mTOR inhibitor in the treatment of cancer, and more particularlyin the treatment of lung cancer, sarcoma, malignant melanoma, prostatecancer, pancreas carcinoma, gastric carcinoma, ovarian cancer, breastcancer, colorectal cancer, kidney cancer and brain cancer. Thischemotherapeutic group includes, but is not limited to sirolimus,temsirolimus, everolimus, ridaforolimus, KU-0063794 and WYE-354.Particularly preferred is the combination of PM01183, or apharmaceutically acceptable salt thereof, with sirolimus, temsirolimus,everolimus, ridaforolimus, KU-0063794 and WYE-354, and even morepreferred is the combination with temsirolimus in the treatment of lungcancer, sarcoma, malignant melanoma, prostate cancer, pancreascarcinoma, gastric carcinoma, ovarian cancer, breast cancer, colorectalcancer and brain cancer.

In another preferred embodiment, the invention is directed to thecombination of PM01183, or a pharmaceutically acceptable salt thereof,with a tyrosine kinase inhibitor in the treatment of cancer, and moreparticularly in the treatment of a cancer selected from lung cancer,sarcoma, prostate cancer, pancreas carcinoma, gastric carcinoma, ovariancancer, hepatoma, breast cancer, colorectal cancer, kidney cancer andbrain cancer. This chemotherapeutic group includes, but is not limitedto erlotinib, sorafenib, axitinib, bosutinib, cediranib, crizotinib,dasatinib, gefitinib, imatinib, canertinib, lapatinib, lestaurtinib,neratinib, nilotinib, semaxanib, sunitinib, vatalanib and vandetanib.Particularly preferred is the combination of PM01183, or apharmaceutically acceptable salt thereof, with erlotinib, sorafenib,axitinib, bosutinib, cediranib, crizotinib, dasatinib, gefitinib,imatinib, canertinib, lapatinib, lestaurtinib, neratinib, nilotinib,semaxanib, sunitinib, vatalanib and vandetanib, and even more preferredis the combination with erlotinib in the treatment of cancer, and moreparticularly in the treatment of a cancer selected from lung cancer,sarcoma, prostate cancer, pancreas carcinoma, gastric carcinoma, ovariancancer, hepatoma, breast cancer, colorectal cancer, kidney cancer andbrain cancer.

In another preferred embodiment, the invention is directed to thecombination of PM01183, or a pharmaceutically acceptable salt thereof,with aplidine in the treatment of cancer, and more particularly in thetreatment of a cancer selected from sarcoma, gastric carcinoma, ovariancancer, colorectal cancer, kidney cancer, brain cancer and leukemia.

In another preferred embodiment, the invention is directed to thecombination of PM01183, or a pharmaceutically acceptable salt thereof,with ET-743 (trabectedin) in the treatment of cancer, and moreparticularly in the treatment of a cancer selected from lung cancer,sarcoma, malignant melanoma, prostate cancer, pancreas carcinoma,gastric carcinoma, ovarian cancer, hepatoma, breast cancer, kidneycancer, leukemia and lymphoma.

In another preferred embodiment, the invention is directed to thecombination of PM01183, or a pharmaceutically acceptable salt thereof,with PM02734 in the treatment of cancer, and more particularly in thetreatment of a cancer selected from sarcoma, malignant melanoma,prostate cancer, pancreas carcinoma, gastric carcinoma, ovarian cancer,breast cancer, colorectal cancer, kidney cancer, brain cancer, leukemiaand lymphoma.

PM02734((4S)-MeHex-D-Val-L-Thr-L-Val-D-Val-D-Pro-L-Orn-D-allo-Ile-cyclo(D-allo-Thr-D-allo-Ile-D-Val-L-Phe-Z-Dhb-L-Val))is a synthetic depsipeptide related to the family of kahalalidecompounds, which is currently in clinical trials for the treatment ofcancer. This compound is the subject of WO 2004/035613 and has thefollowing structure:

In another preferred embodiment, the invention is directed to thecombination of PM01183, or a pharmaceutically acceptable salt thereof,with PM00104 in the treatment of cancer, and more particularly in thetreatment of a cancer selected from lung cancer, sarcoma, malignantmelanoma, prostate cancer, pancreas carcinoma, gastric carcinoma,ovarian cancer, hepatoma, breast cancer, kidney cancer, leukemia andlymphoma.

PM00104 is a synthetic alkaloid related to jorumycin and renieramycins,and also to safracin and saframycin compounds, which is currently inclinical trials for the treatment of cancer, and has the followingstructure:

For further details on PM00104 see WO 01/87894.

The invention includes any pharmaceutically acceptable salt of any drugreferred to herein, which can be synthesized from the parent compound byconventional chemical methods as disclosed before.

In one embodiment, the invention relates to synergistic combinationsemploying PM01183, or a pharmaceutically acceptable salt thereof, andanother anticancer drug selected from the list of drugs given above. Anindication of synergism can be obtained by testing the combinations andanalyzing the results, for example by the Chou-Talalay method or by anyother suitable method, such as those provided in the Examples section.

The possible favorable outcomes for synergism include 1) increasing theefficacy of the therapeutic effect, 2) decreasing the dosage butincreasing or maintaining the same efficacy to avoid toxicity, 3)minimizing or slowing down the development of drug resistance, and 4)providing selective synergism against target (or efficacy synergism)versus host (or toxicity antagonism). Accordingly, in a combination oftwo chemotherapeutic agents having synergism, the treatment regimen willbe different of those in which the combination of the two drugs showsonly an additive effect. In this regard, if there is synergism lessdosage of one or both of the agents (compared with the amounts used insingle therapy) may be required to obtain the same or even a greaterefficacy, and the possible toxic side effects may be reduced or evenavoided. Alternatively, if the dosage of both drugs in the combinationis the same as those when given alone (as single agents), an increase inefficacy of the combination can be expected. Therefore, the existence ofsynergism in a given drug combination will modify the length of thetreatment and/or the treatment regimen.

In another embodiment, the invention relates to a method of increasingor potentiating the therapeutic efficacy of an anticancer drug selectedfrom the list of drugs given above in the treatment of cancer, whichcomprises administering to a patient in need thereof a therapeuticallyeffective amount of PM01183, or a pharmaceutically acceptable saltthereof, in conjunction with this other anticancer drug. An indicationof increase or potentiation of the therapeutic efficacy can be obtainedby testing the combinations and analyzing the results, for example thetumor growth inhibition. This tumor growth inhibition can be assessed bycomparing the mean tumor volume of the treatment combining the two drugs(PM01183 and the other drug) with those of the other drug monotherapytreatment. In this regard, increase or potentiation of the therapeuticefficacy is determined when the response of the combination therapy isgreater than the best response of the most active drug administered assingle agent (monotherapy) on the same schedule and dose as used in thecombination therapy. This aspect of the invention is further illustratedin the Examples section, specifically in Examples 13-19.

In another aspect, the invention is directed to the use of PM01183, or apharmaceutically acceptable salt thereof, for the manufacture of amedicament for the treatment of cancer by combination therapy employingPM01183, or a pharmaceutically acceptable salt thereof, with anotheranticancer drug selected from the list of drugs given above.

In a further aspect, the invention is directed to a method for thetreatment of cancer comprising administering to a patient in need ofsuch treatment a therapeutically effective amount of PM01183, orpharmaceutically acceptable salt thereof, in combination with atherapeutically effective amount of another anticancer drug selectedfrom the list of drugs given above.

In another aspect, the invention is directed to PM01183, or apharmaceutically acceptable salt thereof, for use in the treatment ofcancer comprising administering a therapeutically effective amount ofPM01183, or a pharmaceutical acceptable salt thereof, in combinationwith a therapeutically effective amount of another anticancer drugselected from the list of drugs given above.

According to the present invention, PM01183, or a pharmaceuticallyacceptable salt thereof, and the other anticancer drug may be providedin the same medicament or as separate medicaments for administration atthe same time or at different times. Preferably, PM01183, or apharmaceutically acceptable salt thereof, and the other anticancer drugare provided as separate medicaments for administration at differenttimes. When administered separately and at different times, eitherPM01183, or a pharmaceutically acceptable salt thereof, or the otheranticancer drug, may be administered first. In addition, both drugs canbe administered in the same day or at different days, and they can beadministered using the same schedule or at different schedules duringthe treatment cycle. Additionally, the administration of both drugs canbe done by using the same route of administration or different routes.For instance, both drugs can be administered by intravenousadministration or, alternatively, one drug can be administered orallyand the other one by intravenous administration.

Thus, the pharmaceutical compositions of the present invention maycomprise all the components (drugs) in a single pharmaceuticallyacceptable formulation or, alternatively, the components may beformulated separately and administered in combination with one another.Various pharmaceutically acceptable formulations well known to those ofskill in the art can be used in the present invention. Moreover,selection of an appropriate formulation for use in the present inventioncan be performed by those skilled in the art by taking into account theroute of administration and the solubility characteristics of thecomponents of the composition.

The correct dosage of both drugs in combination will vary according tothe particular formulation, the mode of application, and the particularsite, patient and tumour being treated. Other factors like age, bodyweight, sex, diet, time of administration, rate of excretion, conditionof the patient, other drug combinations, reaction sensitivities andseverity of the disease shall be taken into account. Administration canbe carried out continuously or periodically within the maximum tolerateddose.

The combination of the invention may be used alone or in combinationwith one or more of a variety of anticancer agents or supportive careagents.

In addition, depending on the type of tumor and the development stage ofthe disease, anticancer effects of the treatments of the presentinvention include, but are not limited to, inhibition of tumor growth,tumor growth delay, regression of tumor, shrinkage of tumor, increasedtime to regrowth of tumor on cessation of treatment, slowing of diseaseprogression, and prevention of metastasis. It is expected that when atreatment of the present invention is administered to a patient, such asa human patient, in need of such treatment, said treatment will producean effect, as measured by, for example, the extent of the anticancereffect, the response rate, the time to disease progression, or thesurvival rate. In particular, the treatments of the invention are suitedfor human patients, especially those who are relapsing or refractory toprevious chemotherapy. First line therapy is also envisaged.

In another aspect, the present invention is directed to a kit for use inthe treatment of cancer, comprising a supply of PM01183, or apharmaceutically acceptable salt thereof, in dosage units for at leastone cycle, and printed instructions for the use of PM01183, or apharmaceutically acceptable salt thereof, with another anticancer drugselected from the list of drugs given above in combination.

In a related aspect, the present invention is directed to a kit for usein the treatment of cancer, comprising a supply of PM01183, or apharmaceutically acceptable salt thereof, in dosage units for at leastone cycle, a supply of another anticancer drug selected from the list ofdrugs given above in dosage units for at least one cycle, and printedinstructions for the use of both drugs in combination.

In another aspect, the present invention also provides a pharmaceuticalcomposition comprising PM01183, or a pharmaceutically acceptable saltthereof, and a pharmaceutically acceptable carrier or excipient, for usein combination with another anticancer drug selected from the list ofdrugs given above in the treatment of cancer.

In a further aspect, the present invention also provides apharmaceutical composition comprising PM01183, or a pharmaceuticallyacceptable salt thereof, another anticancer drug selected from the listof drugs given above, and a pharmaceutically acceptable carrier. Thispharmaceutical composition is preferable for use in the treatment ofcancer.

In another aspect, the invention further provides for the use ofPM01183, or a pharmaceutically acceptable salt thereof, in thepreparation of a composition for use in combination with anotheranticancer drug selected from the list of drugs given above in thetreatment of cancer.

In another aspect, the invention further provides for the use ofPM01183, or a pharmaceutically acceptable salt thereof, for thetreatment of cancer, in combination therapy with another anticancer drugselected from the list of drugs given above.

In one embodiment, cancer cells are contacted, or otherwise treated,with a combination of PM01183, or a pharmaceutically acceptable saltthereof, and another anticancer drug selected from the list of drugsgiven above. The cancer cells are preferably human and include carcinomacells, sarcoma cells, leukemia cells, lymphoma cells, and myeloma cells.More preferably, the cancer cells are cells of lung cancer, sarcoma,malignant melanoma, bladder carcinoma, prostate cancer, pancreascarcinoma, thyroid cancer, gastric carcinoma, ovarian cancer, hepatoma,breast cancer, colorectal cancer, kidney cancer, esophageal cancer,neuroblastoma, brain cancer, cervical cancer, anal cancer, testicularcancer, leukemia, multiple myeloma and lymphoma. In addition, thecombination provides a synergistic inhibitory effect against the cancercells, particularly against the human cancer cells mentioned above.

For example, the combination inhibits proliferation or survival ofcontacted cancer cells. A lower level of proliferation or survival ofthe contacted cancer cells compared to the non-contacted cancer cellssupports the combination of PM01183, or a pharmaceutically acceptablesalt thereof, and another anticancer drug selected from the list ofdrugs given above as being effective for treating a patient with cancer.

In another aspect, the invention provides for a method for inhibitingthe growth of cancer cells comprising contacting said cancer cells withan effective amount of PM01183, or a pharmaceutically acceptable saltthereof, in combination with another anticancer drug selected from thelist of drugs given above.

In another aspect, the invention provides for a method for inhibitingthe growth of cancer cells comprising contacting said cancer cells witha synergistic combination of PM01183, or a pharmaceutically acceptablesalt thereof, and another anticancer drug selected from the list ofdrugs given above, wherein said combination provides improved inhibitionagainst cancer cell growth as compared to (i) PM01183, or apharmaceutically acceptable salt thereof, in the absence of the otheranticancer drug, or (ii) the other anticancer drug in the absence ofPM01183.

In another aspect, the invention provides for a pharmaceuticalcomposition comprising a synergistic combination of PM01183, or apharmaceutically acceptable salt thereof, and another anticancer drugselected from the list of drugs given above for inhibiting the growth ofcancer cells, wherein said combination provides improved inhibitionagainst cancer cell growth as compared to (i) PM01183, or apharmaceutically acceptable salt thereof, in the absence of the otheranticancer drug, or (ii) the other anticancer drug in the absence ofPM01183.

In another embodiment, the combination of PM01183, or a pharmaceuticallyacceptable salt thereof, and another anticancer drug selected from thelist of drugs given above inhibits tumor growth or reduces the size of atumor in vivo. In particular, the combination inhibits in vivo growthand/or reduces the size of carcinoma, sarcoma, leukemia, lymphoma, andmyeloma. Preferably, the combination inhibits in vivo tumor growth oflung, sarcoma, malignant melanoma, bladder, prostate, pancreas, thyroid,gastric, ovarian, hepatoma, breast, colorectal, kidney, esophageal,neuroblastoma, brain, cervical, anal, testicular, leukemia, multiplemyeloma and lymphoma tumours.

For example, these combinations inhibit tumor growth or reduce the sizeof human cancer xenografts, particularly human gastric, pancreas,sarcoma, lung, colorectal and ovary tumors xenografts, in animal models.A reduced growth or reduced size of human cancer xenografts in animalmodels administered with these combinations further supports thecombination of PM01183, or a pharmaceutically acceptable salt thereof,and another anticancer drug selected from the list of drugs given aboveas being effective for treating a patient with cancer.

Therefore, in another aspect, the invention provides for a method forreducing the size of a tumor, comprising administering an effectiveamount of PM01183, or a pharmaceutically acceptable salt thereof, incombination with another anticancer drug selected from the list of drugsgiven above.

In another aspect, the invention provides for a method for inhibitingtumor growth, comprising administering an effective amount of PM01183,or a pharmaceutically acceptable salt thereof, in combination withanother anticancer drug selected from the list of drugs given above.

The following examples further illustrate the invention. These examplesshould not be interpreted as a limitation of the scope of the invention.

To provide a more concise description, some of the quantitativeexpressions given herein are not qualified with the term “about”. It isunderstood that, whether the term “about” is used explicitly or not,every quantity given herein is meant to refer to the actual given value,and it is also meant to refer to the approximation to such given valuethat would reasonably be inferred based on the ordinary skill in theart, including equivalents and approximations due to the experimentaland/or measurement conditions for such given value.

EXAMPLES Example 1. In Vitro Studies to Determine the Effect of PM01183in Combination with Chemotherapeutic Agents on Human Lung Carcinoma CellLines

The objective of this study was to determine the ability of PM01183 topotentiate the antitumor activity of chemotherapeutic agents used in thetreatment of lung carcinoma.

The following agents were evaluated in combination with PM01183:oxaliplatin, carmustine, cyclophosphamide, mytomicin C (stock solutionsof these compounds prepared in sterile double distilled water and storedat −20° C.), 5-fluorouracil (5-FU), gemcitabine, paclitaxel, docetaxel,vincristine, daunorubicin, actinomycin D, topotecan, etoposide,bortezomib, vorinostat, dacarbazine, temsirolimus, erlotinib, ET-743 andPM00104 (stock solutions of these compounds prepared in pure DMSO andstored at −20° C.). Additional serial dilutions were prepared inserum-free culture medium to achieve a final 4× concentration. Aliquotsof 50 μL of each diluted compound were added per well.

A549 was the human lung carcinoma cell line selected for this assay.A549 cells were maintained in Dulbecco's modified Eagle's medium (DMEM)supplemented with 10% Fetal Bovine Serum (FBS), 2 mM L-glutamine and 100units/mL of Penicillin-Streptomycin, at 37° C., 5% CO2 and 95% humidity.The screening was performed in two parts:a. In the first set of assays, IC₅₀ values were determined for each drugin A549 cells after 72 hours of drug exposure. Briefly, cells wereharvested and seeded in 96 well microtiter plates at a density of 5,000cells in 150 μL of culture medium and incubated for 24 hours indrug-free medium before treatment with vehicle alone or test compoundsfor 72 h.The cytotoxic effect was measured by the MTT reduction assay, in which3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, atetrazole, which is reduced to purple formazan in the mitochondria ofliving cells, was used. MTT (50 μL of 1 mg/mL stock solution) was addedto the wells and incubated for 8 hours at 37° C. until formazan crystalswere formed. After gently removing the culture medium, DMSO was added todissolve the insoluble purple formazan product into a colored solution.The absorbance of the wells was quantified by measuring the opticaldensity at 540 nm. Results were expressed as percentage of control cellgrowth. The 1050 values (concentration of drug that produces a 50%inhibition of cell growth) used for the combination studies werecalculated using Prism v5.02 software (GraphPad). The results wereexpressed as molar concentration and represented the average of 2-4independent assays.

The IC₅₀ values (72 hours drug exposure) of each individual agent forthe A549 tumor cell line are shown in table 1.

TABLE 1 IC₅₀ values in molar concentration (M) for each of the agentCompound IC₅₀ (M) Compound IC₅₀ (M) Compound IC₅₀ (M) PM01183 3.60E−09Oxaliplatin 9.00E−04 5-FU 9.23E−05 Gemcitabine 2.80E−10 Paclitaxel4.00E−08 Docetaxel 3.00E−09 Vincristine 2.50E−07 Daunorubicin 3.55E−07Mitomycin C 2.49E−04 Actinomycin D 4.70E−09 Topotecan 8.00E−07 Etoposide7.82E−07 Bortezomib 3.10E−09 Vorinostat 6.81E−06 Cyclophosphamide1.00E−03 Carmustine 1.00E−03 Dacarbazine 6.00E−04 Temsirolimus 3.29E−06Erlotinib 1.00E−05 ET-743 2.25E−08 PM00104 7.00E−09b. In a second set of assays, A549 human tumor cells were incubated withPM01183 in combination with each of the agents mentioned above. Thepreviously obtained IC₅₀ values were used as starting concentrations foreach compound (100% concentration). Arbitrary dilutions, as percentageof the initial IC₅₀ value (100%, 75%, 70%, 60%, 50%, 40%, 30%, 25%, and0%), were performed for each pair of compounds and tested in combinedcomplementary (opposite concentrations) dose-response curves as follows:

IC₅₀ of PM01183 IC₅₀ of Agent 100%   0% 75% 25% 70% 30% 60% 40% 50% 50%40% 60% 30% 70% 25% 75%  0% 100% 

As a visual aid, response values were plotted on a scatter plot withdose ratios given on the x-axis and % response values on the y-axis. Ahorizontal line was drawn between the two endpoint response values (E.g.between the response values for 100% IC₅₀ PM01183 and 100% IC₅₀ standardchemotherapeutic agent). In cases where response values at the twoendpoints were approximately equivalent, points lying above or belowthis predicted line of additivity could be interpreted as representingantagonistic or synergistic drug interaction, respectively.

The in vitro combinations of each drug with PM01183 have the potentialto be synergistic, additive or antagonistic. Synergistic cytotoxicity totumor cells is an optimal effect and implies that the combination ofPM01183 with another drug is more effective than either drug alone.

According to this assay, it was found that in A549 human lung carcinomacell line:

a. The combination of PM01183 with oxaliplatin exhibited strongsynergism (FIG. 1).b. The combination of PM0183 with 5-fluorouracil (FIG. 2) and PM01183with gemcitabine (FIG. 3) showed synergism at almost all dose ratios.c. The combination of PM01183 with paclitaxel showed synergism (FIG. 4)at the 50/50-40/60 dose ratios, while the combination of PM01183 withdocetaxel showed synergism (FIG. 5) at the 75/25 and 50/50 dose ratios,and the combination of PM01183 with vincristine exhibited synergism(FIG. 6) at almost all dose ratios.d. The combination of PM01183 with daunorubicin (FIG. 7), PM01183 withmitomycin C (FIG. 8), and PM01183 with actinomycin D (FIG. 9) exhibitedsynergism at almost all dose ratios.e. The combination of PM01183 with topotecan showed strong synergism(FIG. 10), while the combination of PM01183 with etoposide showedsynergism (FIG. 11) at the 60/40 and 25/75 dose ratios.f. The combination of PM01183 with bortezomib showed synergism (FIG. 12)at the 40/60-30/70 dose ratios.g. The combination of PM01183 with vorinostat (FIG. 13) showed strongsynergism at almost all dose ratios.h. The combination of PM01183 with cyclophosphamide (FIG. 14) showedsynergism at almost all dose ratios.i. The combination of PM01183 with carmustine exhibited strong synergism(FIG. 15).j. The combination of PM01183 with dacarbazine showed strong synergism(FIG. 16).k. The combinations of PM01183 with temsirolimus showed synergism (FIG.17) at almost all dose ratios.l. The combination of PM01183 with erlotinib showed strong synergism(FIG. 18).m. The combination of PM01183 with ET-743 showed synergism (FIG. 19) atthe 75/25-60/40 and 30/70 dose ratios.n. The combination of PM01183 with PM00104 (FIG. 20) showed synergism atalmost all dose ratios.

Example 2. In Vitro Studies to Determine the Effect of PM01183 inCombination with Chemotherapeutic Agents on Human Sarcoma Cell Lines

The objective of this study was to determine the ability of PM01183 topotentiate the antitumor activity of chemotherapeutic agents used in thetreatment of sarcoma.

The following agents were evaluated in combination with PM01183:cisplatin, oxaliplatin, cyclophosphamide, mytomicin C (stock solutionsof these compounds prepared in sterile double distilled water and storedat −20° C.), gemcitabine, docetaxel, vincristine, vinorelbine,daunorubicin, cytarabine, actinomycin D, topotecan, etoposide,vorinostat, dacarbazine, temsirolimus, erlotinib, aplidine, PM02734,ET-743 and PM00104 (stock solutions of these compounds prepared in pureDMSO and stored at −20° C.). Additional serial dilutions were preparedin serum-free culture medium to achieve a final 4× concentration.Aliquots of 50 μL of each diluted compound were added per well.

A673 was the human rhabdomyosarcoma cell line selected for this assay.A673 cells were maintained in Dulbecco's modified Eagle's medium (DMEM)supplemented with 10% Fetal Bovine Serum (FBS), 2 mM L-glutamine and 100units/mL of Penicillin-Streptomycin, at 37° C., 5% CO2 and 95% humidity.

The screening was performed in two parts as disclosed in example 1:

a. In the first set of assays, IC₅₀ values were determined for each drugafter 72 hours of drug exposure in the A673 tumor cell line.

The IC₅₀ values (72 hours drug exposure) of each individual agent forthe A673 tumor cell line were calculated by using the same methodologydisclosed in example 1 and are shown in table 2.

TABLE 2 IC₅₀ values in molar concentration (M) for each of the agentCompound IC₅₀ (M) Compound IC₅₀ (M) Compound IC₅₀ (M) PM01183 2.20E−09Cisplatin 3.03-05 Oxaliplatin 7.80E−05 Cytarabine 1.97E−07 Gemcitabine4.34E−10 Docetaxel 6.50E−10 Vincristine 8.60E−09 Vinorelbine 5.00E−08Daunorubicin 5.20E−07 Mitomycin C 2.99E−06 Actinomycin D 9.56E−10Topotecan 2.40E−08 Etoposide 1.55E−06 Vorinostat 2.16E−06Cyclophosphamide 1.00E−03 Dacarbazine 3.00E−04 Temsirolimus 1.00E−06Erlotinib 5.00E−05 Aplidine 2.16E−09 ET-743 1.90E−09 PM02734 3.60E−06PM00104 3.00E−09b. In a second set of assays, A673 human tumor cells were incubated withPM01183 in combination with each of the agents mentioned above in thesame combination of unique IC₅₀ concentrations as those described inexample 1.Cell culture and cell plating were performed as described before and thecytotoxic effect was measured by the MTT Assay as disclosed in example1.

According to this assay, it was found that in A673 human sarcoma cellline:

a. The combination of PM01183 with cisplatin (FIG. 21) and PM01183 withoxaliplatin (FIG. 22) exhibited strong synergism.b. The combination of PM01183 with cytarabine exhibited strong synergism(FIG. 23), while the combination of PM01183 with gemcitabine showedsynergism (FIG. 24) at the 75/25-70/30 dose ratios.c. The combination of PM01183 with docetaxel (FIG. 25), PM01183 withvincristine (FIG. 26) and PM01183 with vinorelbine (FIG. 27) showedsynergism at almost all dose ratios.d. The combination of PM01183 with daunorubicin (FIG. 28) and PM01183with actinomycin D (FIG. 30) showed synergism at almost all dose ratios,while the combination of PM01183 with mitomycin C (FIG. 29) exhibitedstrong synergism.e. The combination of PM01183 with topotecan (FIG. 31) and PM01183 withetoposide (FIG. 32) exhibited strong synergism at almost all doseratios.f. The combination of PM01183 with vorinostat (FIG. 33) showed strongsynergism.g. The combination of PM01183 with cyclophosphamide (FIG. 34) showedsynergism at almost all dose ratios.h. The combination of PM01183 with dacarbazine showed synergism (FIG.35) at the 75/25-70/30 and 40/60 dose ratios.i. The combinations of PM01183 with temsirolimus showed strong synergism(FIG. 36).j. The combination of PM01183 with erlotinib exhibited strong synergism(FIG. 37).k. The combination of PM01183 with aplidine showed synergism (FIG. 38)at the 50/50-30/70 dose ratios.l. The combination of PM01183 with ET-743 (FIG. 39) showed synergism atthe 30/70-25/75 dose ratios.m. The combination of PM01183 with PM02734 (FIG. 40) showed synergism atthe 75/25 and 40/60 dose ratios.n. The combination of PM01183 with PM00104 exhibited synergism (FIG.41).

Example 3. In Vitro Studies to Determine the Effect of PM01183 inCombination with Chemotherapeutic Agents on Human Malignant MelanomaCell Lines

The objective of this study was to determine the ability of PM01183 topotentiate the antitumor activity of chemotherapeutic agents used in thetreatment of malignant melanoma.

The following agents were evaluated in combination with PM01183:cisplatin, mytomicin C (stock solutions of these compounds prepared insterile double distilled water and stored at −20° C.), 5-fluorouracil,doxorubicin, daunorubicin, cytarabine, topotecan, irinotecan,methotrexate, etoposide, dacarbazine, temsirolimus, PM02734, ET-743 andPM00104 (stock solutions of these compounds prepared in pure DMSO andstored at −20° C.). Additional serial dilutions were prepared inserum-free culture medium to achieve a final 4× concentration. Aliquotsof 50 μL of each diluted compound were added per well.

SK-MEL-2 was the human melanoma cell line selected for this assay.SK-MEL-2 cells were maintained in Minimum Essential Medium Eagle (MEME)supplemented with 10% Fetal Bovine Serum (FBS), 2 mM L-glutamine and 100units/mL of Penicillin-Streptomycin, at 37° C., 5% CO2 and 95% humidity.

The screening was performed in two parts as disclosed in example 1:

a. In the first set of assays, IC₅₀ values were determined for each drugafter 72 hours of drug exposure in the SK-MEL-2 tumor cell line.

The IC₅₀ values (72 hours drug exposure) of each individual agent forthe SK-MEL-2 tumor cell line were calculated by using the samemethodology disclosed in example 1 and are shown in table 3.

TABLE 3 IC₅₀ values in molar concentration (M) for each of the agentCompound IC₅₀ (M) Compound IC₅₀ (M) Compound IC₅₀ (M) PM01183 2.00E−09Cisplatin 1.60E−04 5-FU 7.00E−04 Cytarabine 3.89E−06 Methotrexate1.00E−04 Daunorubicin 1.77E−07 Doxorubicin 3.00E−07 Mitomycin C 9.00E−07Topotecan 4.37E−07 Irinotecan 1.80E−05 Etoposide 2.89E−06 Dacarbazine6.30E−04 Temsirolimus 5.00E−05 ET-743 2.00E−09 PM02734 1.76E−06 PM001042.00E−09b. In a second set of assays, SK-MEL-2 tumor cells were incubated withPM01183 in combination with each of the agents mentioned above in thesame combination of unique IC₅₀ concentrations as those described inexample 1.Cell culture and cell plating were performed as described before and thecytotoxic effect was measured by the MTT Assay as disclosed in example1.

According to this assay, it was found that in SK-MEL-2 human melanomacell line:

a. The combination of PM01183 with cisplatin (FIG. 42) showed synergismat the 75/25, 50/50 and 30/70 dose ratios.b. The combination of PM01183 with 5-fluorouracil (FIG. 43), PM01183with cytarabine (FIG. 44), and PM01183 with methotrexate (FIG. 45)exhibited strong synergism.c. The combination of PM01183 with daunorubicin (FIG. 46) and PM01183with doxorubicin (FIG. 47) showed synergism at almost all dose ratios,while the combination of PM01183 with mitomycin C (FIG. 48) exhibitedstrong synergism.d. The combination of PM01183 with topotecan (FIG. 49), PM01183 withirinotecan (FIG. 50), and PM01183 with etoposide (FIG. 51) exhibitedsynergism and even strong synergism in some dose ratios.e. The combination of PM01183 with dacarbazine showed synergism (FIG.52).f. The combinations of PM01183 with temsirolimus showed strong synergism(FIG. 53).g. The combination of PM01183 with ET-743 (FIG. 54) showed synergism atalmost all dose ratios.h. The combination of PM01183 with PM02734 (FIG. 55) showed synergism atthe 25/75-50/50 dose ratios.i. The combination of PM01183 with PM00104 (FIG. 56) exhibited synergismat almost all dose ratios.

Example 4. In Vitro Studies to Determine the Effect of PM01183 inCombination with Chemotherapeutic Agents on Human Prostate CarcinomaCell Lines

The objective of this study was to determine the ability of PM01183 topotentiate the antitumor activity of chemotherapeutic agents used in thetreatment of prostate cancer.

The following agents were evaluated in combination with PM01183:cisplatin, oxaliplatin, mytomicin C (stock solutions of these compoundsprepared in sterile double distilled water and stored at −20° C.),5-fluorouracil, gemcitabine, docetaxel, paclitaxel, vinorelbine,daunorubicin, cytarabine, doxorubicin, actinomycin D, topotecan,irinotecan, methotrexate, etoposide, vorinostat, temsirolimus,bortezomib, erlotinib, flutamide, PM02734, ET-743 and PM00104 (stocksolutions of these compounds prepared in pure DMSO and stored at −20°C.). Additional serial dilutions were prepared in serum-free culturemedium to achieve a final 4× concentration. Aliquots of 50 μL of eachdiluted compound were added per well.

PC-3 was the human prostate adenocarcinome cell line selected for thisassay. PC-3 cells were maintained in Roswell Park Memorial Institutemedium (RPMI) supplemented with 10% Fetal Bovine Serum (FBS), 2 mML-glutamine and 100 units/mL of Penicillin-Streptomycin, at 37° C., 5%CO2 and 95% humidity.

The screening was performed in two parts as disclosed in example 1:

a. In the first set of assays, IC₅₀ values were determined for each drugafter 72 hours of drug exposure in the PC-3 tumor cell line.The IC₅₀ values (72 hours drug exposure) of each individual agent forthe PC-3 tumor cell line were calculated by using the same methodologydisclosed in example 1 and are shown in table 4.

TABLE 4 IC₅₀ values in molar concentration (M) for each of the agentCompound IC₅₀ (M) Compound IC₅₀ (M) Compound IC₅₀ (M) PM01183 2.60E−09Cisplatin 1.10E−04 Oxaliplatin 1.71E−04 5-FU 1.00E−03 Cytarabine4.00E−05 Gemcitabine 4.00E−07 Methotrexate 1.20E−04 Docetaxel 1.86E−08Paclitaxel 9.00E−08 Vinorelbine 1.00E−05 Daunorubicin 1.15E−06Doxorubicin 1.48E−06 Mitomycin C 1.00E−05 Actinomycin D 1.00E−08Topotecan 6.33E−07 Irinotecan 7.00E−05 Etoposide 4.80E−05 Bortezomib8.00E−07 Vorinostat 3.90E−06 Flutamide 4.90E−05 Temsirolimus 5.00E−07Erlotinib 2.33E−04 ET-743 8.00E−09 PM02734 5.40E−07 PM00104 7.10E−09b. In a second set of assays, PC-3 human tumor cells were incubated withPM01183 in combination with each of the agents mentioned above in thesame combination of unique IC₅₀ concentrations as those described inexample 1.Cell culture and cell plating were performed as described before and thecytotoxic effect was measured by the MTT Assay as disclosed in examples1.

According to this assay it was found that in PC-3 human prostate cancercell line:

a. The combination of PM01183 with cisplatin (FIG. 57) showed synergismat almost all dose ratios, while the combination of PM01183 withoxaliplatin (FIG. 58) exhibited strong synergism.b. The combination of PM01183 with 5-fluorouracil (FIG. 59) and PM01183with cytarabine (FIG. 60) exhibited synergism at almost all dose ratios,and the combination of PM01183 with gemcitabine exhibited strongsynergism (FIG. 61). Finally, the combination of PM01183 withmethotrexate showed synergism (FIG. 62) at the 30/70-25/75 dose ratios.c. The combination of PM01183 with docetaxel showed synergism (FIG. 63)at almost all dose ratios, while the combination of PM01183 withpaclitaxel (FIG. 64) showed synergism at the 40/60-30/70 dose ratios.The combination of PM01183 with vinorelbine (FIG. 65) showed strongsynergism.d. The combination of PM01183 with daunorubicin (FIG. 66) and PM01183with doxorubicin (FIG. 67) exhibited strong synergism. The combinationof PM01183 with mitomycin C (FIG. 68) and PM01183 with actinomycin D(FIG. 69) showed synergism at almost all dose ratios.e. The combination of PM01183 with topotecan (FIG. 70) and PM01183 withirinotecan (FIG. 71) exhibited strong synergism, while the combinationof PM01183 with etoposide (FIG. 72) showed synergism at almost all doseratios.f. The combination of PM01183 with bortezomib (FIG. 73) showed synergismat almost all dose ratios.g. The combination of PM01183 with vorinostat (FIG. 74) showedsynergism.h. The combination of PM01183 with flutamide (FIG. 75) showed synergismat the 40/60-25/75 dose ratios.i. The combination of PM01183 with temsirolimus exhibited strongsynergism (FIG. 76).j. The combination of PM01183 with erlotinib (FIG. 77) showed synergismat almost all dose ratios.k. The combination of PM01183 with ET-743 (FIG. 78) showed synergism atalmost all dose ratios.l. The combination of PM01183 with PM02734 (FIG. 79) showed synergism atthe 75/25-70/30 and 30/70 dose ratios.m. The combination of PM01183 with PM00104 exhibited strong synergism(FIG. 80).

Example 5. In Vitro Studies to Determine the Effect of PM01183 inCombination with Chemotherapeutic Agents on Human Pancreas CarcinomaCell Lines

The objective of this study was to determine the ability of PM01183 topotentiate the antitumor activity of chemotherapeutic agents used in thetreatment of pancreatic carcinoma.

The following agents were evaluated in combination with PM01183:cisplatin, oxaliplatin, (stock solutions of these compounds prepared insterile double distilled water and stored at −20° C.), gemcitabine,daunorubicin, cytarabine, doxorubicin, actinomycin D, topotecan,irinotecan, methotrexate, etoposide, vorinostat, temsirolimus,bortezomib, erlotinib, PM02734, ET-743 and PM00104 (stock solutions ofthese compounds prepared in pure DMSO and stored at −20° C.). Additionalserial dilutions were prepared in serum-free culture medium to achieve afinal 4× concentration. Aliquots of 50 μL of each diluted compound wereadded per well.

PANC-1 was the human pancreatic carcinoma cell line selected for thisassay. PANC-1 cells were maintained in Roswell Park Memorial Institutemedium (RPMI) supplemented with 10% Fetal Bovine Serum (FBS), 2 mML-glutamine and 100 units/mL of Penicillin-Streptomycin, at 37° C., 5%CO2 and 95% humidity.

The screening was performed in two parts as disclosed in example 1:

a. In the first set of assays, IC₅₀ values were determined for each drugafter 72 hours of drug exposure in the PANC-1 tumor cell line.The IC₅₀ values (72 hours drug exposure) of each individual agent forthe PANC-1 tumor cell line were calculated by using the same methodologydisclosed in example 1 and are shown in table 5.

TABLE 5 IC₅₀ values in molar concentration (M) for each of the agentCompound IC₅₀ (M) Compound IC₅₀ (M) Compound IC₅₀ (M) PM01183 2.80E−09Cisplatin 1.47E−04 Oxaliplatin 1.84E−04 Cytarabine 9.00E−05 Gemcitabine1.00E−06 Methotrexate 1.00E−05 Daunorubicin 8.69E−07 Doxorubicin3.45E−06 Actinomycin D 2.20E−08 Topotecan 4.37E−06 Irinotecan 9.00E−05Etoposide 1.00E−05 Bortezomib 4.16E−07 Vorinostat 6.05E−06 Temsirolimus1.00E−05 Erlotinib 4.16E−07 ET-743 2.10E−08 PM02734 9.00E−06 PM001047.89E−09b. In a second set of assays, PANC-1 human tumor cells were incubatedwith PM01183 in combination with each of the agents mentioned above inthe same combination of unique IC₅₀ concentrations as those described inexample 1.Cell culture and cell plating were performed as described before and thecytotoxic effect was measured by the MTT Assay as disclosed example 1.

According to this assay it was found that in PANC-1 human pancreascarcinoma cell line:

a. The combination of PM01183 with cisplatin (FIG. 81) and PM01183 withoxaliplatin (FIG. 82) exhibited strong synergism.b. The combination of PM01183 with cytarabine (FIG. 83) showed synergismat almost all dose ratios, while the combination of PM01183 withgemcitabine (FIG. 84) and PM01183 with methotrexate (FIG. 85) exhibitedstrong synergism.c. The combination of PM01183 with daunorubicin (FIG. 86) and PM01183with doxorubicin (FIG. 87) exhibited synergism, while the combination ofPM01183 with actinomycin D (FIG. 88) showed synergism at the 75/25 and30/70-25/75 dose ratios.d. The combination of PM01183 with topotecan (FIG. 89) and PM01183 withirinotecan (FIG. 90) exhibited strong synergism, while the combinationof PM01183 with etoposide (FIG. 91) showed synergism at almost all doseratios.e. The combination of PM01183 with bortezomib (FIG. 92) showed synergismat the 75/25-70/30 and 50/50 dose ratios.f. The combination of PM01183 with vorinostat (FIG. 93) showed synergismat almost all dose ratios.g. The combination of PM01183 with temsirolimus exhibited strongsynergism (FIG. 94).h. The combination of PM01183 with erlotinib exhibited strong synergism(FIG. 95).i. The combination of PM01183 with ET-743 (FIG. 96) showed synergism atalmost all dose ratios.j. The combination of PM01183 with PM02734 (FIG. 97) showed synergism atalmost all dose ratios.k. The combination of PM01183 with PM00104 showed synergism (FIG. 98) atthe 75/25 and 50/50 dose ratios.

Example 6. In Vitro Studies to Determine the Effect of PM01183 inCombination with Chemotherapeutic Agents on Human Gastric Carcinoma CellLines

The objective of this study was to determine the ability of PM01183 topotentiate the antitumor activity of chemotherapeutic agents used in thetreatment of gastric cancer.

The following agents were evaluated in combination with PM01183:cisplatin, oxaliplatin, cyclophosphamide (stock solutions of thesecompounds prepared in sterile double distilled water and stored at −20°C.), 5-fluorouracil, gemcitabine, paclitaxel, vincristine, vinorelbine,daunorubicin, dacarbazine, cytarabine, doxorubicin, actinomycin D,topotecan, irinotecan, methotrexate, etoposide, vorinostat,temsirolimus, bortezomib, erlotinib, aplidine, PM02734, ET-743 andPM00104 (stock solutions of these compounds prepared in pure DMSO andstored at −20° C.). Additional serial dilutions were prepared inserum-free culture medium to achieve a final 4× concentration. Aliquotsof 50 μL of each diluted compound were added per well.

HGC-27 was the human gastric carcinoma cell line selected for thisassay. HGC-27 cells were maintained in Iscove's modified Dulbeco'smedium (IDMD) supplemented with 10% Fetal Bovine Serum (FBS), 2 mML-glutamine and 100 units/mL of Penicillin-Streptomycin, at 37° C., 5%CO2 and 95% humidity.

The screening was performed in two parts as disclosed in example 1:

a. In the first set of assays, IC₅₀ values were determined for each drugafter 72 hours of drug exposure in the HGC-27 tumor cell line.

The IC₅₀ values (72 hours drug exposure) of each individual agent forthe HGC-27 tumor cell line were calculated by using the same methodologydisclosed in example 1 and are shown in table 6.

TABLE 6 IC₅₀ values in molar concentration (M) for each of the agentCompound IC₅₀ (M) Compound IC₅₀ (M) Compound IC₅₀ (M) PM01183 8.50E−10Cisplatin 8.00E−05 Oxaliplatin 1.06E−04 5-FU 1.00E−05 Cytarabine5.00E−05 Gemcitabine 5.34E−10 Methotrexate 3.30E−08 Paclitaxel 5.00E−09Vincristine 1.25E−08 Vinorelbine 6.50E−08 Daunorubicin 3.72E−07Doxorubicin 5.40E−08 Actinomycin D 3.74E−09 Topotecan 8.08E−07Irinotecan 4.00E−06 Etoposide 2.90E−06 Bortezomib 5.60E−09 Vorinostat1.20E−06 Cyclophosphamide 1.00E−03 Dacarbazine 3.46E−04 Temsirolimus1.50E−07 Erlotinib 7.50E−06 Aplidine 9.00E−09 ET-743 5.80E−09 PM027349.50E−07 PM00104 3.20E−09b. In a second set of assays, HGC-27 human tumor cells were incubatedwith PM01183 in combination with each of the agents mentioned above inthe same combination of unique IC₅₀ concentrations as those described inexample 1.Cell culture and cell plating were performed, as described before andthe cytotoxic effect was measured by the MTT Assay, as disclosed inexample 1.

According to this assay it was found that in HGC-27 human gastriccarcinoma cell line:

a. The combination of PM01183 with cisplatin (FIG. 99) showed synergismat almost all dose ratios, while the combination of PM01183 withoxaliplatin (FIG. 100) exhibited strong synergism.b. The combination of PM01183 with 5-fluorouracil (FIG. 101) and PM01183with cytarabine (FIG. 102) exhibited synergism, even being strong insome dose ratios. The combination of PM01183 with gemcitabine (FIG. 103)and PM01183 with methotrexate (FIG. 104) showed synergism at almost alldose ratios.c. The combination of PM01183 with paclitaxel exhibited strong synergism(FIG. 105). The combination of PM01183 with vincristine (FIG. 106) andPM01183 with vinorelbine (FIG. 107) showed synergism at almost all doseratios.d. The combination of PM01183 with daunorubicin (FIG. 108) and PM01183with actinomycin D (FIG. 110) exhibited strong synergism. Thecombination of PM01183 with doxorubicin (FIG. 109) exhibited synergismat the 75/25-60/40 dose ratios.e. The combination of PM01183 with topotecan exhibited strong synergism(FIG. 111). The combination of PM01183 with irinotecan (FIG. 112) showedsynergism at the 70/30-60/40 and 40/60 dose ratios, while thecombination of PM01183 with etoposide (FIG. 113) showed synergism atalmost all dose ratios.f. The combination of PM01183 with bortezomib exhibited strong synergism(FIG. 114).g. The combination of PM01183 with vorinostat (FIG. 115) showedsynergism at almost all dose ratios.h. The combination of PM01183 with cyclophosphamide exhibited strongsynergism (FIG. 116).i. The combination of PM01183 with dacarbazine exhibited strongsynergism (FIG. 117).j. The combination of PM01183 with temsirolimus exhibited strongsynergism (FIG. 118).k. The combination of PM01183 with erlotinib exhibited strong synergism(FIG. 119).l. The combination of PM01183 with aplidine showed strong synergism(FIG. 120).m. The combination of PM01183 with ET-743 (FIG. 121) showed synergism atthe 50/50 and 75/25 dose ratios.n. The combination of PM01183 with PM02734 exhibited strong synergism(FIG. 122).o. The combination of PM01183 with PM00104 (FIG. 123) showed synergismat almost all dose ratios.

Example 7. In Vitro Studies to Determine the Effect of PM01183 inCombination with Chemotherapeutic Agents on Human Ovarian Carcinoma CellLines

The objective of this study was to determine the ability of PM01183 topotentiate the antitumor activity of chemotherapeutic agents used in thetreatment of ovarian cancer.

The following agents were evaluated in combination with PM01183:cisplatin, oxaliplatin, cyclophosphamide, carmustine, mytomicin C (stocksolutions of these compounds prepared in sterile double distilled waterand stored at −20° C.), 5-fluorouracil, gemcitabine, docetaxel,paclitaxel, vincristine, vinorelbine, daunorubicin, dacarbazine,cytarabine, doxorubicin, actinomycin D, topotecan, irinotecan,methotrexate, etoposide, vorinostat, temsirolimus, erlotinib, aplidine,PM02734, ET-743 and PM00104 (stock solutions of these compounds preparedin pure DMSO and stored at −20° C.). Additional serial dilutions wereprepared in serum-free culture medium to achieve a final 4×concentration. Aliquots of 50 μL of each diluted compound were added perwell.

IGROV-1 was the human ovarian adenocarcinoma cell line selected for thisassay. IGROV-1 cells were maintained in Roswell Park Memorial Institutemedium (RPMI) supplemented with 10% Fetal Bovine Serum (FBS), 2 mML-glutamine and 100 units/mL of Penicillin-Streptomycin, at 37° C., 5%CO2 and 95% humidity.

The screening was performed in two parts as disclosed in example 1:

a. In the first set of assays, IC₅₀ values were determined for each drugafter 72 hours of drug exposure in the IGROV-1 tumor cell line.

The IC₅₀ values (72 hours drug exposure) of each individual agent forthe IGROV-1 tumor cell line were calculated by using the samemethodology disclosed in example 1 and are shown in table 7.

TABLE 7 IC₅₀ values in molar concentration (M) for each of the agentCompound IC₅₀ (M) Compound IC₅₀ (M) Compound IC₅₀ (M) PM01183 3.20E−09Cisplatin 7.00E−05 Oxaliplatin 8.50E−06 5-FU 9.00E−05 Cytarabine1.17E−05 Gemcitabine 6.34E−09 Methotrexate 1.00E−04 Docetaxel 5.01E−08Paclitaxel 9.50E−08 Vincristine 3.79E−07 Vinorelbine 1.39E−06Daunorubicin 3.55E−07 Doxorubicin 2.59E−07 Actinomycin D 3.29E−09Mitomycin C 3.00E−06 Topotecan 3.00E−07 Irinotecan 1.00E−05 Etoposide3.06E−06 Vorinostat 2.88E−06 Carmustine 7.12E−04 Cyclophosphamide1.00E−03 Dacarbazine 3.98E−04 Temsirolimus 1.27E−07 Erlotinib 7.91E−06Aplidine 1.50E−09 ET-743 6.45E−09 PM02734 3.33E−07 PM00104 3.30E−09b. In a second set of assays, IGROV-1 human tumor cells were incubatedwith PM01183 in combination with each of the agents mentioned above inthe same combination of unique IC₅₀ concentrations as those described inexample 1.Cell culture and cell plating were performed as described before and thecytotoxic effect was measured by the MTT Assay as disclosed in example1.

According to this assay it was found that in IGROV-1 human ovariancarcinoma cell line:

a. The combination of PM01183 with cisplatin (FIG. 124) showed synergismat almost all dose ratios, while the combination of PM01183 withoxaliplatin exhibited strong synergism (FIG. 125).b. The combination of PM01183 with 5-fluorouracil (FIG. 126) and PM01183with cytarabine (FIG. 127) showed synergism at almost all dose ratios.The combination of PM01183 with gemcitabine (FIG. 128) and PM01183 withmethotrexate (FIG. 129) exhibited synergism.c. The combination of PM01183 with docetaxel (FIG. 130), PM01183 withpaclitaxel (FIG. 131), and PM01183 with vincristine (FIG. 132) exhibitedstrong synergism, while the combination of PM01183 with vinorelbine(FIG. 133) showed synergism at almost all dose ratios.d. The combination of PM01183 with daunorubicin (FIG. 134) exhibitedsynergism. The combination of PM01183 with doxorubicin (FIG. 135) andPM01183 with actinomycin D (FIG. 136) exhibited synergism at almost alldose ratios, while the combination of PM01183 with mitomycin C (FIG.137) showed synergism at the 50/50 and 30/70-25/75 dose ratios.e. The combination of PM01183 with topotecan (FIG. 138), PM01183 withirinotecan (FIG. 139), and PM01183 with etoposide (FIG. 140) exhibitedsynergism.f. The combination of PM01183 with vorinostat (FIG. 141) showedsynergism at almost all dose ratios.g. The combination of PM01183 with cyclophosphamide (FIG. 142) showedsynergism at almost all dose ratios.h. The combination of PM01183 with carmustine (FIG. 143) exhibitedsynergism at almost all dose ratios.i. The combination of PM01183 with dacarbazine (FIG. 144) showedsynergism at almost all dose ratios.j. The combination of PM01183 with temsirolimus exhibited synergism(FIG. 145).k. The combination of PM01183 with erlotinib exhibited synergism (FIG.146).l. The combination of PM01183 with aplidine (FIG. 147) showed synergismat the 70/30-60/40 dose ratios.m. The combination of PM01183 with ET-743 (FIG. 148) showed synergism atthe 75/25-60/40 dose ratios.n. The combination of PM01183 with PM02734 exhibited strong synergism(FIG. 149).o. The combination of PM01183 with PM00104 (FIG. 150) showed synergismat almost all dose ratios.

Example 8. In Vitro Studies to Determine the Effect of PM01183 inCombination with Chemotherapeutic Agents on Human HepatocellularCarcinoma Cell Lines

The objective of this study was to determine the ability of PM01183 topotentiate the antitumor activity of chemotherapeutic agents used in thetreatment of hepatocellular cancer.

The following agents were evaluated in combination with PM01183:cisplatin, oxaliplatin, cyclophosphamide (stock solutions of thesecompounds prepared in sterile double distilled water and stored at −20°C.), 5-fluorouracil, gemcitabine, paclitaxel, docetaxel, vincristine,vinorelbine, daunorubicin, cytarabine, doxorubicin, topotecan,irinotecan, methotrexate, etoposide, bortezomib, erlotinib, ET-743 andPM00104 (stock solutions of these compounds prepared in pure DMSO andstored at −20° C.). Additional serial dilutions were prepared inserum-free culture medium to achieve a final 4× concentration. Aliquotsof 50 μL of each diluted compound were added per well.

HepG2 was the human hepatocellular liver carcinoma cell line selectedfor this assay. HepG2 cells were maintained in Minimum Essential MediumEagle (MEME) supplemented with 10% Fetal Bovine Serum (FBS), 2 mML-glutamine and 100 units/mL of Penicillin-Streptomycin, at 37° C., 5%CO2 and 95% humidity.

The screening was performed in two parts as disclosed in example 1:

a. In the first set of assays, IC₅₀ values were determined for each drugafter 72 hours of drug exposure in the HepG2 tumor cell line.

The IC₅₀ values (72 hours drug exposure) of each individual agent forthe HepG2 tumor cell line were calculated by using the same methodologydisclosed in example 1 and are shown in table 8.

TABLE 8 IC₅₀ values in molar concentration (M) for each of the agentCompound IC₅₀ (M) Compound IC₅₀ (M) Compound IC₅₀ (M) PM01183 2.50E−09Cisplatin 5.00E−05 Oxaliplatin 2.80E−05 5-FU 4.50E−06 Cytarabine2.06E−05 Gemcitabine 5.34E−09 Methotrexate 3.96E−08 Docetaxel 5.00E−07Paclitaxel 5.70E−08 Vincristine 6.00E−08 Vinorelbine 1.02E−06Daunorubicin 3.00E−07 Doxorubicin 2.00E−07 Topotecan 1.00E−06 Irinotecan1.00E−06 Etoposide 1.04E−05 Bortezomib 3.90E−07 Cyclophosphamide1.00E−03 Erlotinib 8.60E−06 ET-743 7.21E−09 PM00104 3.00E−09b. In a second set of assays, HepG2 human tumor cells were incubatedwith PM01183 in combination with each of the agents mentioned above inthe same combination of unique IC₅₀ concentrations as those described inexample 1.Cell culture and cell plating were performed as described before and thecytotoxic effect was measured by the MTT Assay as disclosed in example1.

According to this assay it was found that in HepG2 human hepatocellularcell line:

a. The combination of PM01183 with cisplatin (FIG. 151) and PM01183 withoxaliplatin (FIG. 152) exhibited strong synergism.b. The combination of PM01183 with 5-fluorouracil (FIG. 153) showedsynergism at the 75/25, 50/50 and 30/70 dose ratios. The combination ofPM01183 with cytarabine (FIG. 154), PM01183 with gemcitabine (FIG. 155)and PM01183 with methotrexate (FIG. 156) exhibited strong synergism.c. The combination of PM01183 with docetaxel (FIG. 157) exhibited strongsynergism. The combination of PM01183 with paclitaxel (FIG. 158) andPM01183 with vincristine (FIG. 159) showed synergism at almost all doseratios, while the combination of PM01183 with vinorelbine (FIG. 160)showed synergism at the 50/50 and 30/70-25/75 dose ratios.d. The combination of PM01183 with daunorubicin (FIG. 161) and PM01183with doxorubicin (FIG. 162) showed synergism at almost all dose ratios.e. The combination of PM01183 with topotecan (FIG. 163) and PM01183 withetoposide (FIG. 165) exhibited strong synergism. The combination ofPM01183 with irinotecan (FIG. 164) showed synergism at almost all doseratios.f. The combination of PM01183 with bortezomib (FIG. 166) showedsynergism at the 75/25-60/40 dose ratios.g. The combination of PM01183 with cyclophosphamide (FIG. 167) showedsynergism at almost all dose ratios.h. The combination of PM01183 with erlotinib (FIG. 168) exhibited strongsynergism.i. The combination of PM01183 with ET-743 (FIG. 169) showed synergism atthe 60/40-50/50 dose ratios.j. The combination of PM01183 with PM00104 (FIG. 170) exhibited strongsynergism.

Example 9. In Vitro Studies to Determine the Effect of PM01183 inCombination with Chemotherapeutic Agents on Human Breast Carcinoma CellLines

The objective of this study was to determine the ability of PM01183 topotentiate the antitumor activity of chemotherapeutic agents used in thetreatment of breast cancer.

The following agents were evaluated in combination with PM01183:cisplatin, oxaliplatin, cyclophosphamide, carmustine, mytomicin C (stocksolutions of these compounds prepared in sterile double distilled waterand stored at −20° C.), 5-fluorouracil, gemcitabine, paclitaxel,docetaxel, vincristine, vinorelbine, daunorubicin, dacarbazine,cytarabine, doxorubicin, actinomycin D, topotecan, irinotecan,methotrexate, etoposide, vorinostat, temsirolimus, erlotinib, tamoxifen,PM02734, ET-743 and PM00104 (stock solutions of these compounds preparedin pure DMSO and stored at −20° C.). Additional serial dilutions wereprepared in serum-free culture medium to achieve a final 4×concentration. Aliquots of 50 μL of each diluted compound were added perwell.

MDA-MB-231 was the human breast adenocarcinoma cell line selected forthis assay. MDA-MB-231 cells were maintained in Dulbecco's modifiedEagle's medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS), 2mM L-glutamine and 100 units/mL of Penicillin-Streptomycin, at 37° C.,5% CO2 and 95% humidity.

The screening was performed in two parts as disclosed in example 1:

a. In the first set of assays, IC₅₀ values were determined for each drugafter 72 hours of drug exposure in the MDA-MB-231 tumor cell line.

The IC₅₀ values (72 hours drug exposure) of each individual agent forthe MDA-MB-231 tumor cell line were calculated by using the samemethodology disclosed in example 1 and are shown in table 9.

TABLE 9 IC₅₀ values in molar concentration (M) for each of the agentCompound IC₅₀ (M) Compound IC₅₀ (M) Compound IC₅₀ (M) PM01183 3.50E−09Cisplatin 1.53E−04 Oxaliplatin 1.08E−04 5-FU 9.00E−05 Cytarabine9.57E−06 Gemcitabine 8.50E−09 Methotrexate 5.94E−06 Docetaxel 2.50E−09Paclitaxel 8.50E−09 Vincristine 5.00E−08 Vinorelbine 1.20E−05Daunorubicin 3.70E−07 Doxorubicin 6.00E−07 Actinomycin D 4.54E−10Mitomycin C 2.00E−06 Topotecan 1.66E−07 Irinotecan 8.50E−06 Etoposide4.80E−06 Vorinostat 1.70E−06 Cyclophosphamide 1.00E−03 Carmustine9.00E−04 Dacarbazine 1.92E−05 Tamoxifen 1.30E−05 Temsirolimus 1.20E−05Erlotinib 1.00E−04 ET-743 2.00E−09 PM02734 2.80E−06 PM00104 1.00E−09b. In a second set of assays, MDA-MB-231 human tumor cells wereincubated with PM01183 in combination with each of the agents mentionedabove in the same combination of unique IC₅₀ concentrations as thosedescribed in example 1.Cell culture and cell plating were performed as described before and thecytotoxic effect was measured by the MTT Assay as disclosed in example1.

According to this assay it was found that in MDA-MB-231 human breastcarcinoma cell line:

a. The combination of PM01183 with cisplatin (FIG. 171) and PM01183 withoxaliplatin (FIG. 172) exhibited synergism.b. The combination of PM01183 with 5-fluorouracil (FIG. 173) showedsynergism at almost all dose ratios. The combination of PM01183 withcytarabine (FIG. 174) and PM01183 with gemcitabine (FIG. 175) exhibitedstrong synergism, while the combination of PM01183 with methotrexate(FIG. 176) showed synergism at the 75/25-70/30 and 50/50 dose ratios.c. The combination of PM01183 with docetaxel (FIG. 177) and PM01183 withpaclitaxel (FIG. 178) exhibited synergism. The combination of PM01183with vincristine (FIG. 179) showed synergism at the 75/25 and 50/50 doseratios, while the combination of PM01183 with vinorelbine (FIG. 180)showed synergism at almost all dose ratios.d. The combination of PM01183 with daunorubicin (FIG. 181) and PM01183with mitomycin C (FIG. 184) exhibited synergism at almost all doseratios. The combination of PM01183 with doxorubicin (FIG. 182) exhibitedstrong synergism and the combination of PM01183 with actinomycin D (FIG.183) exhibited synergism.e. The combination of PM01183 with topotecan (FIG. 185) showed synergismat almost all dose ratios. The combination of PM01183 with irinotecan(FIG. 186) and PM01183 with etoposide (FIG. 187) exhibited synergism.f. The combination of PM01183 with vorinostat (FIG. 188) showedsynergism at 75/25 and 50/50-40/60 dose ratios.g. The combination of PM01183 with cyclophosphamide (FIG. 189) exhibitedstrong synergism.h. The combination of PM01183 with carmustine (FIG. 190) exhibitedsynergism at almost all dose ratios.i. The combination of PM01183 with dacarbazine (FIG. 191) showedsynergism at almost all dose ratios.j. The combination of PM01183 with tamoxifen (FIG. 192) showed synergismat almost all dose ratiosk. The combination of PM01183 with temsirolimus exhibited strongsynergism (FIG. 193).l. The combination of PM01183 with erlotinib exhibited strong synergism(FIG. 194).m. The combination of PM01183 with ET-743 exhibited strong synergism(FIG. 195).n. The combination of PM01183 with PM02734 (FIG. 196) exhibitedsynergism at almost all dose ratios.o. The combination of PM01183 with PM00104 (FIG. 197) showed synergismat almost all dose ratios.

Example 10. In Vitro Studies to Determine the Effect of PM01183 inCombination with Chemotherapeutic Agents on Human Colorectal CarcinomaCell Lines

The objective of this study was to determine the ability of PM01183 topotentiate the antitumor activity of chemotherapeutic agents used in thetreatment of colorectal cancer.

The following agents were evaluated in combination with PM01183:cisplatin, oxaliplatin, cyclophosphamide, mytomicin C (stock solutionsof these compounds prepared in sterile double distilled water and storedat −20° C.), 5-fluorouracil, gemcitabine, docetaxel, vinorelbine,daunorubicin, dacarbazine, cytarabine, doxorubicin, actinomycin D,topotecan, irinotecan, etoposide, vorinostat, bortezomib, temsirolimus,erlotinib, PM02734 and aplidine (stock solutions of these compoundsprepared in pure DMSO and stored at −20° C.). Additional serialdilutions were prepared in serum-free culture medium to achieve a final4× concentration. Aliquots of 50 μL of each diluted compound were addedper well.

HT-29 was the human colon adenocarcinoma cell line selected for thisassay. HT-29 cells were maintained in Dulbecco's modified Eagle's medium(DMEM) supplemented with 10% Fetal Bovine Serum (FBS), 2 mM L-glutamineand 100 units/mL of Penicillin-Streptomycin, at 37° C., 5% CO2 and 95%humidity.

The screening was performed in two parts as disclosed in example 1:

a. In the first set of assays, IC₅₀ values were determined for each drugafter 72 hours of drug exposure in the HT-29 tumor cell line.

The IC₅₀ values (72 hours drug exposure) of each individual agent forthe HT-29 tumor cell line were calculated by using the same methodologydisclosed in example 1 and are shown in table 10.

TABLE 10 IC₅₀ values in molar concentration (M) for each of the agentCompound IC₅₀ (M) Compound IC₅₀ (M) Compound IC₅₀ (M) PM01183 3.70E−09Cisplatin 2.20E−04 Oxaliplatin 1.03E−04 5-FU 9.00E−06 Cytarabine7.80E−06 Gemcitabine 4.00E−07 Docetaxel 3.20E−10 Vinorelbine 3.00E−08Daunorubicin 5.32E−07 Doxorubicin 9.00E−07 Actinomycin D 3.27E−09Mitomycin C 2.00E−06 Topotecan 3.28E−07 Irinotecan 9.00E−06 Etoposide5.44E−06 Bortezomib 6.15E−09 Vorinostat 2.76E−06 Cyclophosphamide1.00E−03 Dacarbazine 2.47E−05 Temsirolimus 3.50E−06 Erlotinib 2.56E−05Aplidine 1.76E−09 PM02734 2.14E−07b. In a second set of assays, HT-29 human tumor cells were incubatedwith PM01183 in combination with each of the agents mentioned above inthe same combination of unique IC₅₀ concentrations as those described inexample 1.Cell culture and cell plating were performed as described before and thecytotoxic effect was measured by the MTT Assay as disclosed in example1.

According to this assay it was found that in HT-29 human colorectalcarcinoma cell line:

a. The combination of PM01183 with cisplatin (FIG. 198) showed synergismat the 75/25-70/30 dose ratios, while the combination of PM01183 withoxaliplatin (FIG. 199) exhibited strong synergism.b. The combination of PM01183 with 5-fluorouracil (FIG. 200) and PM01183with gemcitabine (FIG. 202) showed synergism at almost all dose ratios,and the combination of PM01183 with cytarabine (FIG. 201) exhibitedstrong synergism.c. The combination of PM01183 with docetaxel (FIG. 203) exhibitedsynergism at the 50/50 and 75/25 dose ratios, while the combination ofPM01183 with vinorelbine (FIG. 204) showed synergism at almost all doseratios.d. The combination of PM01183 with daunorubicin (FIG. 205) and PM01183with mitomycin C (FIG. 208) exhibited strong synergism. The combinationof PM01183 with doxorubicin (FIG. 206) and PM01183 with actinomycin D(FIG. 207) showed synergism at almost all dose ratios.e. The combination of PM01183 with topotecan (FIG. 209) and PM01183 withetoposide (FIG. 211) exhibited strong synergism. The combination ofPM01183 with irinotecan (FIG. 210) showed synergism at almost all doseratios.f. The combination of PM01183 with bortezomib (FIG. 212) showedsynergism at almost all dose ratios.g. The combination of PM01183 with vorinostat (FIG. 213) exhibitedsynergism.h. The combination of PM01183 with cyclophosphamide (FIG. 214) showedsynergism at the 40/60-25/75 dose ratios.i. The combination of PM01183 with dacarbazine (FIG. 215) exhibitedstrong synergism.j. The combination of PM01183 with temsirolimus exhibited strongsynergism (FIG. 216).k. The combination of PM01183 with erlotinib showed synergism at almostall dose ratios (FIG. 217).l. The combination of PM01183 with aplidine (FIG. 218) showed synergismat the 40/60-25/75 dose ratios.m. The combination of PM01183 with PM02734 (FIG. 219) showed synergismat almost all dose ratios.

Example 11. In Vitro Studies to Determine the Effect of PM01183 inCombination with Chemotherapeutic Agents on Human Kidney Carcinoma CellLines

The objective of this study was to determine the ability of PM01183 topotentiate the antitumor activity of chemotherapeutic agents used in thetreatment of kidney cancer.

The following agents were evaluated in combination with PM01183:cisplatin, cyclophosphamide, mytomicin C (stock solutions of thesecompounds prepared in sterile double distilled water and stored at −20°C.), 5-fluorouracil, gemcitabine, methotrexate, docetaxel, vincristine,vinorelbine, daunorubicin, dacarbazine, cytarabine, doxorubicin,actinomycin D, topotecan, irinotecan, etoposide, vorinostat, erlotinib,PM02734, ET-743, PM00104 and aplidine (stock solutions of thesecompounds prepared in pure DMSO and stored at −20° C.). Additionalserial dilutions were prepared in serum-free culture medium to achieve afinal 4× concentration. Aliquots of 50 μL of each diluted compound wereadded per well.

RXF-393 was the human kidney carcinoma cell line selected for thisassay. RXF-393 cells were maintained in Roswell Park Memorial Institutemedium (RPMI) supplemented with 10% Fetal Bovine Serum (FBS), 2 mML-glutamine and 100 units/mL of Penicillin-Streptomycin, at 37° C., 5%CO2 and 95% humidity.

The screening was performed in two parts as disclosed in example 1:

a. In the first set of assays, IC₅₀ values were determined for each drugafter 72 hours of drug exposure in the RXF-393 tumor cell line.

The IC₅₀ values (72 hours drug exposure) of each individual agent forthe RXF-393 tumor cell line were calculated by using the samemethodology disclosed in example 1 and are shown in table 11.

TABLE 11 IC₅₀ values in molar concentration (M) for each of the agentCompound IC₅₀ (M) Compound IC₅₀ (M) Compound IC₅₀ (M) PM01183 5.00E−09Cisplatin 6.67E−05 5-FU 3.00E−04 Cytarabine 5.00E−05 Gemcitabine5.00E−07 Methotrexate 1.75E−04 Docetaxel 5.94E−10 Vincristine 1.73E−08Vinorelbine 8.50E−06 Daunorubicin 6.20E−07 Doxorubicin 8.00E−07Actinomycin D 7.09E−10 Mitomycin C 9.00E−06 Topotecan 3.93E−07Irinotecan 1.40E−05 Etoposide 2.00E−05 Vorinostat 4.10E−06Cyclophosphamide 1.00E−03 Dacarbazine 7.94E−04 Erlotinib 4.80E−06Aplidine 1.50E−09 ET-743 9.60E−09 PM02734 5.00E−06 PM00104 5.40E−09b. In a second set of assays, RXF-393 human tumor cells were incubatedwith PM01183 in combination with each of the agents mentioned above inthe same combination of unique IC₅₀ concentrations as those described inexample 1.Cell culture and cell plating were performed as described before and thecytotoxic effect was measured by the MTT Assay as disclosed in example1.

According to this assay it was found that in RXF-393 human kidneycarcinoma cell line:

a. The combination of PM01183 with cisplatin (FIG. 220) showed synergismat almost all dose ratios.b. The combination of PM01183 with 5-fluorouracil (FIG. 221), PM01183with cytarabine (FIG. 222), PM01183 with gemcitabine (FIG. 223), andPM01183 with methotrexate (FIG. 224) showed synergism at almost all doseratios.c. The combination of PM01183 with docetaxel (FIG. 225), PM01183 withvincristine (FIG. 226) and PM01183 with vinorelbine (FIG. 227) showedsynergism at almost all dose ratios.d. The combination of PM01183 with daunorubicin (FIG. 228) showedsynergism at almost all dose ratios. The combination of PM01183 withdoxorubicin (FIG. 229) showed synergism at the 75/25-60/40 dose ratios,while the combination of PM01183 with actinomycin D (FIG. 230) showedsynergism at the 75/25-70/30 and 30/70 dose ratios. The combination ofPM01183 with mitomycin C (FIG. 231) exhibited strong synergism.e. The combination of PM01183 with topotecan (FIG. 232) exhibited strongsynergism. The combination of PM01183 with irinotecan (FIG. 233) showedsynergism at almost all dose ratios, while the combination of PM01183with etoposide (FIG. 234) showed synergism at the 75/25 and 40/60-30/70dose ratios.f. The combination of PM01183 with vorinostat (FIG. 235) showedsynergism at almost all dose ratios.g. The combination of PM01183 with cyclophosphamide (FIG. 236) showedsynergism at the 75/25-70/30 and 25/75 dose ratios.h. The combination of PM01183 with dacarbazine (FIG. 237) showedsynergism at the 60/40-50/50 dose ratios.i. The combination of PM01183 with erlotinib exhibited strong synergism(FIG. 238).j. The combination of PM01183 with aplidine (FIG. 239) showed synergismat almost all dose ratios.k. The combination of PM01183 with ET-743 (FIG. 240) showed synergism atalmost all dose ratios.l. The combination of PM01183 with PM02734 (FIG. 241) showed synergismat almost all dose ratios.m. The combination of PM01183 with PM00104 (FIG. 242) exhibited strongsynergism.

Example 12. In Vitro Studies to Determine the Effect of PM01183 inCombination with Chemotherapeutic Agents on Human Glioblastoma CellLines

The objective of this study was to determine the ability of PM01183 topotentiate the antitumor activity of chemotherapeutic agents used in thetreatment of glioblastoma.

The following agents were evaluated in combination with PM01183:cisplatin, oxaliplatin (stock solutions of these compounds prepared insterile double distilled water and stored at −20° C.), 5-fluorouracil,gemcitabine, docetaxel, vincristine, daunorubicin, dacarbazine,doxorubicin, topotecan, irinotecan, methotrexate, etoposide, vorinostat,temsirolimus, bortezomib erlotinib, PM02734, ET-743 and aplidine (stocksolutions of these compounds prepared in pure DMSO and stored at −20°C.). Additional serial dilutions were prepared in serum-free culturemedium to achieve a final 4× concentration. Aliquots of 50 μL of eachdiluted compound were added per well.

U87-MG was the human glioblastoma cell line selected for this assay.U87-MG cells were maintained in Minimum Essential Medium Eagle (MEME)supplemented with 10% Fetal Bovine Serum (FBS), 2 mM L-glutamine and 100units/mL of Penicillin-Streptomycin, at 37° C., 5% CO2 and 95% humidity.

The screening was performed in two parts as disclosed in example 1:

a. In the first set of assays, IC₅₀ values were determined for each drugafter 72 hours of drug exposure in the U87-MG tumor cell line.

The IC₅₀ values (72 hours drug exposure) of each individual agent forthe U87-MG tumor cell line were calculated by using the same methodologydisclosed in example 1 and are shown in table 12.

TABLE 12 IC₅₀ values in molar concentration (M) for each of the agentCompound IC₅₀ (M) Compound IC₅₀ (M) Compound IC₅₀ (M) PM01183 4.50E−09Cisplatin 4.40E−05 Oxaliplatin 1.90E−04 5-FU 1.00E−03 Gemcitabine4.50E−07 Methotrexate 5.00E−05 Docetaxel 1.00E−07 Vincristine 1.00E−07Daunorubicin 2.84E−07 Doxorubicin 3.00E−07 Topotecan 7.50E−07 Irinotecan7.54E−06 Etoposide 1.85E−05 Bortezomib 4.00E−07 Vorinostat 1.60E−05Dacarbazine 7.00E−04 Temsirolimus 3.50E−06 Erlotinib 1.49E−04 Aplidine3.80E−09 ET-743 5.00E−09 PM02734 4.08E−06b. In a second set of assays, U87-MG human tumor cells were incubatedwith PM01183 in combination with each of the agents mentioned above inthe same combination of unique IC₅₀ concentrations as those described inexample 1.Cell culture and cell plating were performed as described before and thecytotoxic effect was also measured by the MTT Assay as disclosed inexample 1.

According to this assay it was found that in U87-MG human glioblastomacell line:

a. The combination of PM01183 with cisplatin (FIG. 243) showed synergismat the 70/30 and 50/50 dose ratios, while the combination of PM01183with oxaliplatin (FIG. 244) exhibited strong synergism.b. The combination of PM01183 with 5-fluorouracil (FIG. 245) and PM01183with methotrexate (FIG. 247) exhibited synergism. The combination ofPM01183 with gemcitabine (FIG. 246) showed synergism at almost all doseratios.c. The combination of PM01183 with docetaxel (FIG. 248) and PM01183 withvincristine (FIG. 249) exhibited strong synergism.d. The combination of PM01183 with daunorubicin (FIG. 250) showedsynergism at almost all dose ratios, while the combination of PM01183with doxorubicin (FIG. 251) showed synergism at the 75/25 and 60/40 doseratios.e. The combination of PM01183 with topotecan (FIG. 252) and PM01183 withetoposide (FIG. 254) showed strong synergism. The combination of PM01183with irinotecan (FIG. 253) showed synergism at almost all dose ratios.f. The combination of PM01183 with bortezomib (FIG. 255) showedsynergism at almost all dose ratios.g. The combination of PM01183 with vorinostat (FIG. 256) exhibitedstrong synergism.h. The combination of PM01183 with dacarbazine (FIG. 257) exhibitedsynergism.i. The combination of PM01183 with temsirolimus (FIG. 258) showedsynergism at the 50/50 and 30/70 dose ratios.j. The combination of PM01183 with erlotinib (FIG. 259) showed synergismat the 40/60-25/75 dose ratios.k. The combination of PM01183 with aplidine (FIG. 260) showed synergismat the 50/50-25/75 dose ratios.m. The combination of PM01183 with ET-743 (FIG. 261) exhibited strongsynergism.l. The combination of PM01183 with PM02734 (FIG. 262) showed strongsynergism.

Example 13. In Vivo Studies to Determine the Effect of PM01183 inCombination with Paclitaxel, Vinorelbine and Doxorubicin in HumanOvarian Tumor Xenografts

The aim of these studies was to evaluate the ability of PM01183 topotentiate the antitumor activity of paclitaxel, vinorelbine anddoxorubicin by using a xenograft model of human ovarian carcinoma.Female athymic nude mice (Harlan Laboratories Models, S.L. (Barcelona,Spain) were utilized for all experiments. Animals were housed inindividually ventilated cages, up to ten per cage in a 12-hourlight-dark cycle at 21-23° C. and 40-60% humidity. The mice were allowedfree access to irradiated standard rodent diet and sterilized water.Animals were acclimated for at least 5 days prior to tumor implantationwith a tumor cell suspension.The tumor model used in these studies was A2780 cell line, which wasobtained from the European Collection of Cell Cultures (ECACC n^(o)93112519).A2780 cells were grown at 37° C. with 5% CO₂ in RPMI-1640 medium. Eachanimal was subcutaneously implanted on the right flank, using 26G needleand a 1 cc syringe, with 1×10⁷ A2780 cells (from in vitro passage 5 inPM01183 and doxorubicin and PM01183 and vinorelbine studies; and passage9 in PM01183 and paclitaxel study), in 0.05 mL suspension of 50%Matrigel and 50% serum free medium, without antibiotics.Tumor measurements were determined by using digital caliper (FowlerSylvac, S235PAT). The formula to calculate volume for a prolateellipsoid was used to estimate tumor volume (mm³) from 2-dimensionaltumor measurements: Tumor volume (mm³)=[L×W²]÷2, where L is the lengthand it is the longest diameter in mm, and W is the width and it is theshortest diameter in mm of a tumor. Assuming unit density, volume wasconverted to weight (i.e., 1 mm³=1 mg). Tumor volume and animal bodyweights were measured 2-3 times per week starting form the first day oftreatment (Day 0).Treatment tolerability was assessed by monitoring body weight evolution,clinical signs as well as evidences of local damage in the injectionsite.When tumors reached a volume of about 195 mm³ in the study of PM01183with paclitaxel, a volume of about 158 mm³ in the study of PM01183 withvinorelbine and a volume of about 163.5 mm³ in the study of PM01183 withdoxorubicin, the mice were randomly allocated into the treatments andcontrol groups (N=5-7/group) based on body weight and tumor volumenmeasurements by using NewLab Oncology Software (version 2.25.06.00).PM01183 was provided in the form of vials of lyophilized PM01183 cakewhich was reconstituted with water for infusion to a concentration of0.2 mg/mL. The PM01183 stock solution was further diluted in 5% glucosesolution for injection to the dosing formulation concentrations.Doxorubicin was provided in the form of a solid powder containingDoxorubicin HCl, which was reconstituted in 0.9% saline solution.Vinorelbine was provided as a solution prepared by diluting the productwith 0.9% saline solution. Paclitaxel was provided in the form of asolution prepared by diluting the product with 5% glucose solution forinjection to the target final concentration.In these experiments, PM01183 and paclitaxel, PM01183 and vinorelbineand PM01183 and doxorubicin treatments, as well as placebo, wereintravenously administered once per week up to 2 consecutive weeks onDays 0 and 7. Dose level groups were administered either as singleagents or in combination.Comparison of the median tumor volume in the treatment groups (T) to themedian tumor volume in the control group (T/C×100%) was used forevaluation of the antitumor efficacy. In addition, potentiation wasdetermined when the response of the combination group was greater thanthe best response of the most active agent administered as single agent(monotherapy) on the same schedule and dose as those used in thecombination therapy.Finally, the combination index (CI), that quantitatively measures thedegree of drug interactions, was obtained from the fractions affected bythe treatment, Fa (defined as 1−T/C) for each experimental group at thelast measurement day (Day 10 for PM01183 and paclitaxel combinationstudy, and PM01183 and doxorubicin study, and Day 9 for PM01183 andvinorelbine study) using the median-effect principle (Chou T. C.Pharmacol. Rev. 2006, 58, 621-681).Table 13 reports the % T/C values obtained with PM01183 and paclitaxelboth administered as single agents and in combination for each doselevel, and FIG. 263 shows the tumor volume evaluation of A2780 tumors inmice treated with placebo, PM01183, paclitaxel, and the correspondingcombinations for the groups dosed at the two highest ratios.

TABLE 13 Test % T/C on day Group Dose materials 0 3 5 7 10 G01 10 ml/kgPlacebo — — — — — (Control group) G02 0.18 mg/kg PM01183 101.6 68.9 83.169.1 52.8 G03 0.135 mg/kg PM01183 101.2 89.9 99.8 84.5 61.2 G04 0.09mg/kg PM01183 94.2 88.5 114.1 103.3 88.0 G05 0.045 mg/kg PM01183 94.091.1 99.6 88.0 73.1 G06 25 mg/kg Paclitaxel 95.3 49.3 42.9 34.0 19.8 G0718.75 mg/kg Paclitaxel 95.0 60.4 43.2 41.5 31.1 G08 12.5 mg/kgPaclitaxel 96.2 62.5 73.9 62.5 50.8 G09 6.25 mg/kg Paclitaxel 94.3 60.279.7 81.3 59.2 G10 0.18 mg/kg PM01183 93.3 45.9 28.8 20.9 9.2 25 mg/kgPaclitaxel G11 0.135 mg/kg PM01183 93.4 40.5 37.1 36.0 22.6 18.75 mg/kgPaclitaxel G12 0.09 mg/kg PM01183 96.5 64.3 67.7 73.2 49.0 12.5 mg/kgPaclitaxel G13 0.045 mg/kg PM01183 96.2 78.6 89.1 91.1 77.2 6.25 mg/kgPaclitaxel Placebo: lyophilised cake containing 100 mg Sucrose +Potassium dihydrogen phosphate 6.8 mg + Phosphoric acid q.s. pH 3.8-4.5,which was reconstituted with 1 mL of water for infusion.Table 14 reports the % T/C values obtained with PM01183 and vinorelbineboth administered as single agents and in combination for each doselevel, and FIG. 264 shows the tumor volume evaluation of A2780 tumors inmice treated with placebo, PM01183, vinorelbine, and the correspondingcombinations for the groups dosed at the two highest ratios.

TABLE 14 Test % T/C on day Group Dose materials 0 2 5 7 9 G01 10 ml/kgPlacebo — — — — — (Control group) G02 0.18 mg/kg PM01183 98.9 101.6 72.261.3 62.8 G03 0.135 mg/kg PM01183 98.3 105.3 77.2 79.1 78.7 G04 0.09mg/kg PM01183 98.0 88.6 61.2 87.6 94.5 G05 0.045 mg/kg PM01183 97.8107.5 93.6 92.5 97.1 G06 16.0 mg/kg Vinorelbine 99.0 62.5 20.8 24.5 20.0G07 12.0 mg/kg Vinorelbine 97.4 67.2 39.1 43.0 36.1 G08 8.0 mg/kgVinorelbine 97.6 79.5 45.0 54.2 47.9 G09 4.0 mg/kg Vinorelbine 97.2 88.669.3 81.7 77.3 G10 0.18 mg/kg PM01183 97.3 50.1 10.9 10.6 8.6 16.0 mg/kgVinorelbine G11 0.135 mg/kg PM01183 97.2 74.0 29.6 31.2 26.8 12.0 mg/kgVinorelbine G12 0.09 mg/kg PM01183 96.8 69.3 48.3 56.5 49.8 8.0 mg/kgVinorelbine G13 0.045 mg/kg PM01183 97.1 85.6 61.7 74.2 81.6 4.0 mg/kgVinorelbine Placebo: as disclosed in table 13.Table 15 reports the % T/C values obtained with PM01183 and doxorubicinboth administered as single agents and in combination for each doselevel, and FIG. 265 shows the tumor volume evaluation of A2780 tumors inmice treated with placebo, PM01183, doxorubicin, and the correspondingcombinations for the groups dosed at the two highest ratios.

TABLE 15 Test % T/C on day Group Dose materials 0 3 5 7 10 G01 10 ml/kgPlacebo — — — — — (Control group) G02 0.18 mg/kg PM01183 100.9 70.2 68.569.3 62.1 G03 0.135 mg/kg PM01183 102.2 82.4 86.6 89.2 82.4 G04 0.09mg/kg PM01183 100.2 93.3 95.2 93.5 87.7 G05 0.045 mg/kg PM01183 100.198.2 98.6 97.7 90.0 G06 8.0 mg/kg Doxorubicin 99.5 60.8 49.8 48.1 39.4G07 6.0 mg/kg Doxorubicin 99.4 71.0 60.3 56.8 54.3 G08 4.0 mg/kgDoxorubicin 102.0 82.9 75.1 75.0 68.9 G09 2.0 mg/kg Doxorubicin 99.891.5 93.1 94.2 86.2 G10 0.18 mg/kg PM01183 99.7 47.6 32.6 30.3 21.1 8.0mg/kg Doxorubicin G11 0.135 mg/kg PM01183 100.6 67.0 54.9 53.9 44.9 6.0mg/kg Doxorubicin G12 0.09 mg/kg PM01183 98.3 74.7 69.0 63.1 64.4 4.0mg/kg Doxorubicin G13 0.045 mg/kg PM01183 98.1 83.1 86.6 78.1 79.2 2.0mg/kg Doxorubicin Placebo: as disclosed in table 13.

According to these assays it was found that:

a. The combination treatment of PM01183 and paclitaxel was effective inthe inhibition of the growth of the A2780 ovarian cells, resulting in astatistically significant (P<0.01) tumor reduction compared to thecontrol group with T/C values of 9.2% and 22.6% (Day 10) in the twohighly-dosed groups. Moreover, the combination of PM01183 and paclitaxelproduced lower T/C values than the more active single agent in thisexperiment (paclitaxel at doses of 25 mg/kg and 18.75 mg/kg).Specifically, the TC (%) values of the combination (25 mg/kgpaclitaxel+0.18 mg/kg PM01183) vs paclitaxel alone (25 mg/kg paclitaxel)were 28.8 vs 42.9 (day 5), 20.9 vs 34.0 (day 7), and 9.2 vs 19.8 (day10), and the TC (%) values of the combination (18.75 mg/kgpaclitaxel+0.135 mg/kg PM01183) vs paclitaxel alone (18.75 mg/kgpaclitaxel) were 37.1 vs 43.2 (day 5), 36.0 vs 41.5 (day 7), and 22.6 vs31.1 (day 10). Therefore, when PM01183 is combined with paclitaxel apotentiation of the antitumor activity is clearly observed.Additionally, based on the median-effect principle, the combination ofPM01183 and paclitaxel resulted in CI values less than 1 (at Fa higherthan 0.8), indicating synergism in mice bearing ovarian A2780xenografted tumors.b. The combination treatment of PM01183 and vinorelbine was effective inthe inhibition of the growth of the A2780 ovarian cells, resulting in astatistically significant (P<0.01) tumor reduction compared to thecontrol group with T/C values of 8.6% and 26.8% (Day 9) in the twohighly-dosed groups. Moreover, the combination of PM01183 andvinorelbine produced lower T/C values than the more active single agentin this experiment (vinorelbine at doses of 16 mg/kg and 12 mg/kg).Specifically, the TC (%) values of the combination (16 mg/kgvinorelbine+0.18 mg/kg PM01183) vs vinorelbine alone (16 mg/kgvinorelbine) were 10.9 vs 20.8 (day 5), 10.6 vs 24.5 (day 7), and 8.6 vs20.0 (day 9), and the TC (%) values of the combination (12 mg/kgvinorelbine+0.135 mg/kg PM01183) vs vinorelbine alone (12 mg/kgvinorelbine) were 29.6 vs 39.1 (day 5), 31.2 vs 43 (day 7), and 26.8 vs36.1 (day 9). Therefore, when PM01183 is combined with vinorelbine apotentiation of the antitumor activity is clearly observed.Additionally, based on the median-effect principle, the combination ofPM01183 and vinorelbine resulted in CI values of 0.75 (at Fa equal to0.97), indicating synergism in mice bearing ovarian A2780 xenograftedtumors.c. The combination treatment of PM01183 and doxorubicin was effective inthe inhibition of the growth of the A2780 ovarian cells, resulting in astatistically significant (P<0.01) tumor reduction compared to thecontrol group with T/C values of 21.1% and 44.9% (Day 10) in the twohighly-dosed groups. Moreover, the combination of PM01183 anddoxorubicin produced lower T/C values than the more active single agentin this experiment (doxorubicin at a dose of 8 mg/kg). Specifically, theTC (%) values of the combination (8 mg/kg doxorubicin+0.18 mg/kgPM01183) vs doxorubicin alone (8 mg/kg doxorubicin) were 32.6 vs 49.8(day 5), 30.3 vs 48.1 (day 7), and 21.1 vs 39.4 (day 10). Therefore,when PM01183 is combined with doxorubicin a potentiation of theantitumor activity is clearly observed.Additionally, based on the median-effect principle, the combination ofPM01183 and doxorubicin resulted in CI values less than 1 (at Fa higherthan 0.8), indicating synergism in mice bearing ovarian A2780xenografted tumors.

Example 14. In Vivo Studies to Determine the Effect of PM01183 inCombination with Cisplatin and 5-Fluorouracil in Human Gastric TumorXenografts

The aim of these studies was to evaluate the ability of PM01183 topotentiate the antitumor activity of cisplatin and 5-fluorouracil byusing a xenograft model of human gastric carcinoma.Female athymic nude mice (Harlan Laboratories Models, S.L. (Barcelona,Spain) were utilized for all experiments. Animals were housed inindividually ventilated cages, up to ten per cage in a 12-hourlight-dark cycle at 21-23° C. and 40-60% humidity. The mice were allowedfree access to irradiated standard rodent diet and sterilized water.Animals were acclimated for at least 5 days prior to tumor implantationwith a tumor cell suspension.The tumor model used in these studies was HGC-27 cell line, which wasobtained from the European Collection of Cell Cultures (ECACC n^(o)94042256).HGC-27 cells were grown at 37° C. with 5% CO₂ in Iscove's modifiedDulbeco's medium (IDMD). Each animal was subcutaneously implanted on theright flank, using 26G needle and a 1 cc syringe, with 5×10⁶ HGC-27cells (from in vitro passage 4 in PM01183 and cisplatin study, andpassage 6 in PM01183 and 5-fluorouracil study), in 0.05 mL suspension of50% Matrigel and 50% serum free medium, without antibiotics.Tumor measurements and treatment tolerability were performed anddetermined as disclosed in Example 13.When tumors reached a volume of about 165.5 mm³ in the study of PM01183with cisplatin and a volume of about 170 mm³ in the study of PM01183with 5-fluorouracil, mice were randomly allocated into the treatmentsand control groups (N=5-7/group) based on body weight and tumor volumenmeasurements by using NewLab Oncology Software (version 2.25.06.00).PM01183 was provided in the form of vials of lyophilized PM01183 cakewhich was reconstituted with water for infusion to a concentration of0.2 mg/mL. The PM01183 stock solution was further diluted in 5% glucosesolution for injection to the dosing formulation concentrations.Cisplatin and 5-fluorouracil were provided as solutions prepared bydiluting the product with 0.9% saline solution for injection to thetarget final concentration.In these experiments, PM01183 and cisplatin and PM01183 and5-fluorouracil treatments, as well as placebo, were intravenouslyadministered once per week up to 2 consecutive weeks on Days 0 and 7.Dose level groups were administered either as single agents or incombination.Comparison of the median tumor volume in the treatment groups (T) to themedian tumor volume in the control group (T/C×100%) was used forevaluation of the antitumor efficacy. In addition, potentiation andcombination index (CI) were determined as disclosed in Example 13.Table 16 reports the % T/C values obtained with PM01183 and cisplatinboth administered as single agents and in combination for each doselevel, and FIG. 266 shows the tumor volume evaluation of HGC-27 tumorsin mice treated with placebo, PM01183, cisplatin, and the correspondingcombinations for the groups dosed at the two highest ratios.

TABLE 16 % T/C on day Group Dose Test materials 0 3 5 7 10 12 14 G01 10ml/kg Placebo — — — — — — — (Control group) G02 0.18 mg/kg PM01183 99.665.9 55.6 38.7 33.5 24.3 24.3 G03 0.135 mg/kg PM01183 97.9 71.6 59.947.8 39.3 37.1 38.3 G04 0.09 mg/kg PM01183 98.6 67.5 67.9 66.1 70.2 60.365.0 G05 0.045 mg/kg PM01183 98.9 85.9 83.1 92.1 76.4 81.6 88.5 G06 6.0mg/kg Cisplatin 97.7 76.1 79.0 75.1 64.4 61.3 72.7 G07 4.5 mg/kgCisplatin 98.5 90.5 94.5 90.2 75.7 73.7 81.1 G08 3.0 mg/kg Cisplatin99.0 78.6 80.0 78.7 81.3 82.8 85.1 G09 1.5 mg/kg Cisplatin 99.3 78.178.8 82.6 83.5 86.6 89.9 G10 0.18 mg/kg PM01183 95.7 55.0 42.4 22.3 12.97.6 4.6 6.0 mg/kg Cisplatin G11 0.135 mg/kg PM01183 99.2 67.7 42.7 28.617.3 12.1 9.8 4.5 mg/kg Cisplatin G12 0.09 mg/kg PM01183 99.9 80.0 64.345.7 47.2 42.4 56.7 3.0 mg/kg Cisplatin G13 0.045 mg/kg PM01183 99.993.3 83.0 75.9 69.3 70.3 80.0 1.5 mg/kg Cisplatin Placebo: as disclosedin table 13.Table 17 reports the % T/C values obtained with PM01183 and5-fluorouracil both administered as single agents and in combination foreach dose level, and FIG. 267 shows the tumor volume evaluation ofHGC-27 tumors in mice treated with placebo, PM01183, 5-fluorouracil, andthe corresponding combinations combinations for the groups dosed at thetwo highest ratios.

TABLE 17 Test % T/C on day Group Dose materials 0 2 5 7 9 12 14 G01 10ml/kg Placebo — — — — — — — (Control group) G02 0.18 mg/kg PM01183 99.678.6 50.9 43.3 41.0 33.0 29.2 G03 0.135 mg/kg PM01183 100.2 81.5 58.761.4 60.2 54.6 55.1 G04 0.09 mg/kg PM01183 100.6 90.5 87.6 83.4 82.676.7 67.7 G05 0.045 mg/kg PM01183 99.9 84.3 103.2 104.6 103.5 101.6 85.0G06 50.0 mg/kg 5-Fluorouracil 100.3 81.2 82.3 81.1 75.6 69.6 60.7 G0737.5 mg/kg 5-Fluorouracil 99.4 86.9 86.9 78.6 73.2 76.7 83.1 G08 25.0mg/kg 5-Fluorouracil 100.6 89.8 97.0 111.4 102.6 93.9 82.8 G09 12.5mg/kg 5-Fluorouracil 100.7 81.7 101.3 102.8 98.6 90.5 83.8 G10 0.18mg/kg PM01183 99.6 73.0 44.2 35.9 31.5 25.3 22.0 50.0 mg/kg5-Fluorouracil G11 0.135 mg/kg PM01183 100.8 73.4 63.5 53.1 50.6 42.851.1 37.5 mg/kg 5-Fluorouracil G12 0.09 mg/kg PM01183 99.6 95.8 97.798.9 90.0 74.7 69.9 25.0 mg/kg 5-Fluorouracil G13 0.045 mg/kg PM0118399.5 80.6 87.3 88.5 99.3 87.1 84.2 12.5 mg/kg 5-Fluorouracil Placebo: asdisclosed in table 13.

According to these assays it was found that:

a. The combination treatment of PM01183 and cisplatin was effective inthe inhibition of the growth of the HGC-27 gastric cells, resulting in astatistically significant (P<0.01) tumor reduction compared to thecontrol group with T/C values of 4.6% and 9.8% (Day 14) in the twohighly-dosed groups. Moreover, the combination of PM01183 and cisplatinproduced lower T/C values than the more active single agent in thisexperiment (PM01183 at doses of 0.18 mg/kg and 0.135 mg/kg).Specifically, the TC (%) values of the combination (6 mg/kgcisplatin+0.18 mg/kg PM01183) vs PM01183 alone (0.18 mg/kg PM01183) were12.9 vs 33.5 (day 10), 7.6 vs 24.3 (day 12), and 4.6 vs 24.3 (day 14),and the TC (%) values of the combination (4.5 mg/kg cisplatin+0.135mg/kg PM01183) vs PM01183 alone (0.135 mg/kg PM01183) were 17.3 vs 39.3(day 10), 12.1 vs 37.1 (day 12), and 9.8 vs 38.3 (day 14). Therefore,when PM01183 is combined with paclitaxel a potentiation of the antitumoractivity is clearly observed.Additionally, based on the median-effect principle, the combination ofPM01183 and cisplatin resulted in CI values less than 1 (at Fa higherthan 0.8), indicating synergism in mice bearing gastric HGC-27xenografted tumors.b. The combination treatment of PM01183 and 5-fluorouracil was effectivein the inhibition of the growth of the HGC-27 gastric cells, resultingin a statistically significant (P<0.01) tumor reduction compared to thecontrol group with T/C values of 22.0% and 51.1% (Day 14) in the twohighly-dosed groups. Moreover, the combination of PM01183 and5-fluorouracil produced lower T/C values than the more active singleagent in this experiment (PM01183 at a dose of 0.18 mg/kg).Specifically, the TC (%) values of the combination (50 mg/kg5-fluorouracil+0.18 mg/kg PM01183) vs PM01183 alone (0.18 mg/kg PM01183)were 35.9 vs 43.3 (day 7), 31.5 vs 41.0 (day 9), 25.3 vs 33.0 (day 12),and 22.0 vs 29.2 (day 14). Therefore, when PM01183 is combined with5-fluorouracil a potentiation of the antitumor activity is clearlyobserved.Additionally, based on the median-effect principle, the combination ofPM01183 and 5-fluorouracil resulted in CI values of 0.78 (at Fa equal to0.97), indicating moderate synergism in mice bearing gastric HGC-27xenografted tumors.

Example 15. In Vivo Studies to Determine the Effect of PM01183 inCombination with Gemcitabine in Human Pancreatic Tumor Xenografts

The aim of these studies was to evaluate the ability of PM01183 topotentiate the antitumor activity of gemcitabine by using a xenograftmodel of human pancreatic cancer.

Female athymic nude mice (Harlan Laboratories Models, S.L. (Barcelona,Spain) were utilized for all experiments. Animals were housed inindividually ventilated cages, up to ten per cage in a 12-hourlight-dark cycle at 21-23° C. and 40-60% humidity. The mice were allowedfree access to irradiated standard rodent diet and sterilized water.Animals were acclimated for at least 5 days prior to tumor implantationwith a tumor cell suspension.The tumor model used in these studies was SW1990 cell line, which wasobtained from the American Type Culture Collection (ATCC: CRL-2172™).SW1990 cells were grown at 37° C. with 5% CO₂ in RPMI-1640 medium. Eachanimal was subcutaneously implanted on the right flank, using 26G needleand a 1 cc syringe, with 5×10⁶ SW1990 cells, from in vitro passage 12,in 0.05 mL suspension of 50% Matrigel and 50% serum free medium, withoutantibiotics.Tumor measurements and treatment tolerability were performed anddetermined as disclosed in Example 13.When tumors reached a volume of about 210 mm³ mice were randomlyallocated into the treatments and control groups (N=5-7/group) based onbody weight and tumor volumen measurements by using NewLab OncologySoftware (version 2.25.06.00).PM01183 was provided in the form of vials of lyophilized PM01183 cakewhich was reconstituted with water for infusion to a concentration of0.2 mg/mL. The PM01183 stock solution was further diluted in 5% glucosesolution for injection to the dosing formulation concentrations.Gemcitabine was provided as a solution prepared by reconstituting theproduct with 0.9% saline solution for injection to a concentration of 40mg/ml stock solution. The gemcitabine stock solution was further dilutedwith 0.9% saline solution for injection to the target finalconcentration.In these experiments, PM01183 and gemcitabine treatment, as well asplacebo, were intravenously administered once per week up to 3consecutive weeks on Days 0, 7 and 14. Dose level groups wereadministered either as single agents or in combination.Comparison of the median tumor volume in the treatment groups (T) to themedian tumor volume in the control group (T/C×100%) was used forevaluation of the antitumor efficacy. In addition, potentiation andcombination index were determined as disclosed in Example 13.Table 18 reports the % T/C values obtained with PM01183 and gemcitabineboth administered as single agents and in combination for each doselevel, and FIG. 268 shows the tumor volume evaluation of SW1990 tumorsin mice treated with placebo, PM01183, gemcitabine, and thecorresponding combinations for the groups dosed at the two highestratios.

TABLE 18 % T/C on day Group Dose Test materials 0 3 6 8 10 13 G01 10ml/kg Placebo — — — — — — (Control group) G02 0.18 mg/kg PM01183 100.074.3 61.3 59.4 56.7 56.1 G03 0.135 mg/kg PM01183 99.6 81.3 71.0 73.165.6 63.1 G04 0.09 mg/kg PM01183 101.1 81.5 72.8 68.7 68.4 74.4 G050.045 mg/kg PM01183 100.2 83.6 82.8 93.3 82.9 88.1 G06 180.0 mg/kgGemcitabine 102.2 84.1 73.9 66.1 60.9 59.4 G07 135.0 mg/kg Gemcitabine102.3 78.3 71.9 63.7 55.4 52.7 G08 90.0 mg/kg Gemcitabine 103.8 70.073.8 63.3 55.6 54.8 G09 45.0 mg/kg Gemcitabine 102.3 85.5 70.3 70.5 63.364.8 G10 0.18 mg/kg PM01183 102.1 69.7 51.2 46.2 36.0 34.1 180.0 mg/kgGemcitabine G11 0.135 mg/kg PM01183 100.4 64.6 52.8 51.5 48.9 46.0 135.0mg/kg Gemcitabine G12 0.09 mg/kg PM01183 98.2 83.2 64.4 59.7 50.6 49.690.0 mg/kg Gemcitabine G13 0.045 mg/kg PM01183 97.7 81.6 70.9 68.8 65.965.7 45.0 mg/kg Gemcitabine % T/C on day Group Dose Test materials 15 1720 22 24 28 G01 10 ml/kg Placebo — — — — — — (Control group) G02 0.18mg/kg PM01183 53.2 47.8 44.2 45.3 44.8 38.9 G03 0.135 mg/kg PM01183 56.356.7 56.9 56.5 53.0 51.7 G04 0.09 mg/kg PM01183 74.7 80.7 71.9 75.4 77.363.9 G05 0.045 mg/kg PM01183 92.6 86.5 85.1 84.5 85.8 85.4 G06 180.0mg/kg Gemcitabine 58.5 52.1 49.1 48.6 46.9 39.3 G07 135.0 mg/kgGemcitabine 54.8 51.2 49.5 48.7 49.8 49.5 G08 90.0 mg/kg Gemcitabine49.9 47.4 47.6 47.0 45.9 49.2 G09 45.0 mg/kg Gemcitabine 63.1 58.5 58.757.3 65.2 59.3 G10 0.18 mg/kg PM01183 34.7 31.6 31.7 28.0 26.0 22.7180.0 mg/kg Gemcitabine G11 0.135 mg/kg PM01183 42.4 38.2 36.6 34.6 31.525.8 135.0 mg/kg Gemcitabine G12 0.09 mg/kg PM01183 47.4 46.0 43.8 49.146.0 42.9 90.0 mg/kg Gemcitabine G13 0.045 mg/kg PM01183 57.9 59.9 55.954.9 52.1 50.5 45.0 mg/kg Gemcitabine Placebo: as disclosed in table 13.

According to this assay it was found that:

a. The combination treatment of PM01183 and gemcitabine was effective inthe inhibition of the growth of the SW 1990 pancreatic cells, resultingin a statistically significant (P<0.01) tumor reduction compared to thecontrol group with T/C values of 22.7% and 25.8% (Day 28) in the twohighly-dosed groups. Moreover, the combination of PM01183 andgemcitabine produced lower T/C values than the more active single agentin this experiment (PM01183 at a dose of 0.18 mg/kg). Specifically, theTC (%) values of the combination (180 mg/kg gemcitabine+0.18 mg/kgPM01183) vs PM01183 alone (0.18 mg/kg PM01183) were 31.7 vs 44.2 (day20), 28.0 vs 45.3 (day 22), 26.0 vs 44.8 (day 24), and 22.7 vs 38.9 (day28). Therefore, when PM01183 is combined with gemcitabine a potentiationof the antitumor activity is clearly observed.Additionally, based on the median-effect principle, the combination ofPM01183 and gemcitabine resulted in CI values less than 1 (at Fa higherthan 0.8), indicating synergism in mice bearing pancreatic SW 1990xenografted tumors.

Example 16. In Vivo Studies to Determine the Effect of PM01183 inCombination with Temozolomide in Human Brain Tumor Xenografts

The aim of these studies was to evaluate the ability of PM01183 topotentiate the antitumor activity of temozolomide by using a xenograftmodel of human brain tumor.Female athymic nude mice (Harlan Laboratories Models, S.L. (Barcelona,Spain) were utilized for all experiments. Animals were housed inindividually ventilated cages, up to ten per cage in a 12-hourlight-dark cycle at 21-23° C. and 40-60% humidity. The mice were allowedfree access to irradiated standard rodent diet and sterilized water.Animals were acclimated for at least 5 days prior to tumor implantationwith a tumor cell suspension.The tumor model used in these studies was U87-MG cell line, which wasobtained from the American Type Culture Collection (ATCC HTB-14™).U87-MG cells were grown at 37° C. with 5% CO₂ in Minimum EssentialMedium Eagle (MEME). Each animal was subcutaneously implanted on theright flank, using 26G needle and a 1 cc syringe, with 5×10⁶ U87-MGcells, from in vitro passage 5, in 0.05 mL suspension of 50% Matrigeland 50% serum free medium, without antibiotics.Tumor measurements and treatment tolerability were performed anddetermined as disclosed in Example 13.When tumors reached a volume of about 139 mm³, mice were randomlyallocated into the treatments and control groups (N=5-7/group) based onbody weight and tumor volumen measurements by using NewLab OncologySoftware (version 2.25.06.00).PM01183 was provided in the form of vials of lyophilized PM01183 cakewhich was reconstituted with water for infusion to a concentration of0.2 mg/mL. The PM01183 stock solution was further diluted in 5% glucosesolution for injection to the dosing formulation concentrations.Temozolomide was provided as a solution prepared by diluting the productin DMSO 10% in 0.9% saline solution for injection to the target finalconcentration.In these experiments, PM01183 and temozolomide treatments, as well asplacebo, were administered as follows: PM01183, intravenously once perweek up to 3 consecutive weeks, on Days 0, 7 and 14, temozolomideorally, in a daily basis during 8 consecutive days (Days 0 to 7), andplacebo was administered following the same schedule as those providedfor PM01183 and temozolomide. Dose level groups were administered eitheras single agents or in combination.Comparison of the median tumor volume in the treatment groups (T) to themedian tumor volume in the control group (T/C×100%) was used forevaluation of the antitumor efficacy. In addition, potentiation andcombination index (CI) were determined as disclosed in Example 13.Table 19 reports the % T/C values obtained with PM01183 and temozolomideboth administered as single agents and in combination for each doselevel, and FIG. 269 shows the tumor volume evaluation of U87-MG tumorsin mice treated with placebo, PM01183, temozolomide, and thecorresponding combinations for the groups dosed at the two highestratios.

TABLE 19 Test % T/C on day Group Dose materials 0 2 4 7 9 11 14 16 G0110 ml/kg Placebo — — — — — — — — (Control group) G02 0.18 mg/kg PM0118399.8 95.5 64.8 63.2 52.0 44.1 38.5 37.1 G03 0.135 mg/kg PM01183 98.590.5 61.2 71.3 67.7 65.3 64.2 63.6 G04 0.09 mg/kg PM01183 97.9 99.5 74.485.1 69.4 71.8 74.1 73.5 G05 0.045 mg/kg PM01183 98.2 101.0 80.4 83.878.8 77.7 76.7 82.5 G06 3.0 mg/kg Temozolomide 97.1 95.5 67.3 39.4 25.322.9 28.4 31.5 G07 1.5 mg/kg Temozolomide 94.1 96.9 75.6 73.0 56.5 59.350.0 53.5 G08 1.0 mg/kg Temozolomide 98.2 100.2 65.1 81.2 55.0 63.5 73.175.0 G09 0.75 mg/kg Temozolomide 97.7 98.9 76.3 77.3 64.4 63.1 62.8 72.7G10 0.18 mg/kg PM01183 97.8 95.0 50.9 33.1 21.0 18.3 16.6 17.4 3.0 mg/kgTemozolomide G11 0.135 mg/kg PM01183 98.7 102.4 62.7 42.0 30.3 29.1 29.030.9 1.5 mg/kg Temozolomide G12 0.09 mg/kg PM01183 96.2 101.0 79.3 76.149.8 51.2 57.6 56.5 1.0 mg/kg Temozolomide G13 0.045 mg/kg PM01183 101106.0 67.4 73.0 57.8 59.0 69.3 72.2 0.75 mg/kg Temozolomide Placebo: asdisclosed in table 13.

According to this assay it was found that:

a. The combination treatment of PM01183 and temozolomide was effectivein the inhibition of the growth of the U87-MG brain tumor cells,resulting in a statistically significant (P<0.01) tumor reductioncompared to the control group with T/C values of 17.4% and 30.9% (Day16) in the two highly-dosed groups. Moreover, the combination of PM01183and temozolomide produced lower T/C values than the more active singleagent in this experiment (temozolomide at doses of 3 mg/kg and 1.5mg/kg).Specifically, the TC (%) values of the combination (3 mg/kgtemozolomide+0.18 mg/kg PM01183) vs temozolomide alone (3 mg/kgtemozolomide) were 18.3 vs 22.9 (day 11), 16.6 vs 28.4 (day 14), and17.4 vs 31.5 (day 16), and the TC (%) values of the combination (1.5mg/kg temozolomide+0.135 mg/kg PM01183) vs temozolomide alone (1.5 mg/kgtemozolomide) were 29.1 vs 59.3 (day 11), 29.0 vs 50.0 (day 14), and30.9 vs 53.5 (day 16). Therefore, when PM01183 is combined withtemozolomide a potentiation of the antitumor activity is clearlyobserved.Additionally, based on the median-effect principle, the combination ofPM01183 and temozolomide resulted in CI values less than 1 (at Fa higherthan 0.8), indicating synergism in mice bearing brain U87-MG xenograftedtumors.

Example 17. In Vivo Studies to Determine the Effect of PM01183 inCombination with Irinotecan in Human Lung Tumor Xenografts

The aim of these studies was to evaluate the ability of PM01183 topotentiate the antitumor activity of iriniotecan by using a xenograftmodel of human lung cancer.Female athymic nude mice (Harlan Laboratories Models, S.L. (Barcelona,Spain) were utilized for all experiments. Animals were housed inindividually ventilated cages, up to ten per cage in a 12-hourlight-dark cycle at 21-23° C. and 40-60% humidity. The mice were allowedfree access to irradiated standard rodent diet and sterilized water.Animals were acclimated for at least 5 days prior to tumor implantationwith a tumor cell suspension.The tumor model used in these studies was H460 cell line, which wasobtained from the American Type Culture Collection of Cell Cultures(ATCC ref. HTB-177™).H460 cells were grown at 37° C. with 5% CO₂ in Dulbecco's modifiedEagle's medium (DMEM). Each animal was subcutaneously implanted on theright flank, using 26G needle and a 1 cc syringe, with 5×10⁶ H460 cells,from in vitro passage 10, in 0.05 mL suspension of 50% Matrigel and 50%serum free medium, without antibiotics.Tumor measurements and treatment tolerability were performed anddetermined as disclosed in Example 13.When tumors reached a volume of about 177 mm³, mice were randomlyallocated into the treatments and control groups (N=5-7/group) based onbody weight and tumor volumen measurements by using NewLab OncologySoftware (version 2.25.06.00).PM01183 was provided in the form of vials of lyophilized PM01183 cakewhich was reconstituted with water for infusion to a concentration of0.2 mg/mL. The PM01183 stock solution was further diluted in 5% glucosesolution for injection to the dosing formulation concentrations.Irinotecan was provided in the form of a solution prepared by dilutingthe product with 5% glucose solution for injection to the target finalconcentration.In these experiments, PM01183 and irinotecan treatments, as well asplacebo, were intravenously administered as follows: PM01183 once perweek up to 2 consecutive weeks, on Days 0 and 7, irinotecan was dosedevery 4 days, on Days 0, 4 and 8, and placebo was administered followingthe same schedule as those provided for PM01183 and irinotecan. Doselevel groups were administered either as single agents or incombination.Comparison of the median tumor volume in the treatment groups (T) to themedian tumor volume in the control group (T/C×100%) was used forevaluation of the antitumor efficacy. In addition, potentiation andcombination index (CI) were determined as disclosed in Example 13.Table 20 reports the % T/C values obtained with PM01183 and irinotecanboth administered as single agents and in combination for each doselevel, and FIG. 270 shows the tumor volume evaluation of H460 tumors inmice treated with placebo, PM01183, irinotecan, and the correspondingcombinations for the groups dosed at the two highest ratios.

TABLE 20 Test % T/C on day Group Dose materials 0 2 5 7 9 12 G01 10ml/kg Placebo — — — — — — (Control group) G02 0.18 mg/kg PM01183 114.479.6 74.7 75.0 69.1 64.9 G03 0.135 mg/kg PM01183 117.6 77.4 67.5 71.766.7 52.9 G04 0.09 mg/kg PM01183 116.9 83.1 83.9 76.9 80.6 84.9 G050.045 mg/kg PM01183 108.3 78.7 61.2 67.2 78.8 87.9 G06 50.0 mg/kgIrinotecan 112.1 54.9 34.7 27.5 24.8 22.9 G07 37.5 mg/kg Irinotecan114.9 51.9 44.0 36.7 35.6 37.0 G08 25.0 mg/kg Irinotecan 112.0 55.6 54.949.6 53.1 51.8 G09 12.5 mg/kg Irinotecan 97.5 50.3 44.4 48.6 50.0 51.5G10 0.18 mg/kg PM01183 117.1 44.3 19.4 13.4 10.9 9.0 50.0 mg/kgIrinotecan G11 0.135 mg/kg PM01183 111.2 51.7 23.8 18.4 15.7 15.3 37.5mg/kg Irinotecan G12 0.09 mg/kg PM01183 110.0 53.2 38.1 26.6 28.0 27.125.0 mg/kg Irinotecan G13 0.045 mg/kg PM01183 109.0 60.4 60.1 56.5 60.058.5 12.5 mg/kg Irinotecan Placebo: as disclosed in table 13.

According to this assay it was found that:

a. The combination treatment of PM01183 and irinotecan was effective inthe inhibition of the growth of the H460 lung cells, resulting in astatistically significant (P<0.01) tumor reduction compared to thecontrol group with T/C values of 9.0% and 15.3% (Day 12) in the twohighly-dosed groups. Moreover, the combination of PM01183 and irinotecanproduced lower T/C values than the more active single agent in thisexperiment (irinotecan at doses of 50 mg/kg and 37.5 mg/kg).Specifically, the TC (%) values of the combination (50 mg/kgirinotecan+0.18 mg/kg PM01183) vs irinotecan alone (50 mg/kg irinotecan)were 19.4 vs 34.7 (day 5), 13.4 vs 27.5 (day 7), 10.9 vs 24.8 (day 9),and 9.0 vs 22.9 (day 12), and the TC (%) values of the combination (37.5mg/kg irinotecan+0.135 mg/kg PM01183) vs irinotecan alone (37.5 mg/kgirinotecan) were 23.8 vs 44.0 (day 5), 18.4 vs 36.7 (day 7), 15.7 vs35.6 (day 9), and 15.3 vs 37.0 (day 12). Therefore, when PM01183 iscombined with irinotecan a potentiation of the antitumor activity isclearly observed.Additionally, based on the median-effect principle, the combination ofPM01183 and irinotecan resulted in CI values less than 1 (at Fa higherthan 0.8), indicating synergism or strong synergism in mice bearing lungH460 xenografted tumors.

Example 18. In Vivo Studies to Determine the Effect of PM01183 inCombination with Dacarbazine in Human Fibrosarcoma Xenografts

The aim of these studies was to evaluate the ability of PM01183 topotentiate the antitumor activity of temozolomide by using a xenograftmodel of human fibrosarcoma.Female athymic nude mice (Harlan Laboratories Models, S.L. (Barcelona,Spain) were utilized for all experiments. Animals were housed inindividually ventilated cages, up to ten per cage in a 12-hourlight-dark cycle at 21-23° C. and 40-60% humidity. The mice were allowedfree access to irradiated standard rodent diet and sterilized water.Animals were acclimated for at least 5 days prior to tumor implantationwith a tumor cell suspension.The tumor model used in these studies was HT1080 cell line, which wasobtained from the American Type Culture Collection (ATCC CCL-121™).HT1080 cells were grown at 37° C. with 5% CO₂ in Minimum EssentialMedium Eagle (MEME). Each animal was orthotopically implanted intogastroecnemius muscle by an intramuscular injection using 26G needle anda 1 cc syringe, with 5×10⁶ HT1080 cells, from in vitro passage 9,suspended in serum free medium, without antibiotics.Total diameter (tumor+leg) measurements were determined by using digitalcaliper (Fowler Sylvac, S235PAT). This total diameter and animal bodyweights were measured 2-3 times per week starting from the first day oftreatment.Treatment tolerability was assessed by monitoring body weight evolution,clinical signs as well as evidences of local damage in the injectionsite.When total diameter reached a length of about 11.3 mm, mice wererandomly allocated into the treatments and control groups (N=5-7/group)based on body weight and tumor measurements by using NewLab OncologySoftware (version 2.25.06.00).PM01183 was provided in the form of vials of lyophilized PM01183 cakewhich was reconstituted with water for infusion to a concentration of0.2 mg/mL. The PM01183 stock solution was further diluted in 5% glucosesolution for injection to the dosing formulation concentrations.Dacarbazine was provided in the form of a solution prepared by dilutingthe product with 5% glucose solution for injection to the target finalconcentration.In these experiments, PM01183 and dacarbazine treatments, as well asplacebo, were intravenously administered once per week up to 2consecutive weeks, on Days 0 and 7. Dose level groups were administeredeither as single agents or in combination.Comparison of the median total diameter (tumor+leg) in the treatmentgroups (T) to the median total diameter (tumor+leg) in the control group(T/C×100%) was used for evaluation of the antitumor efficacy. Inaddition, potentiation and combination index (CI) were determined asdisclosed in Example 13.Table 21 reports the % T/C values obtained with PM01183 and dacarbazineboth administered as single agents and in combination for each doselevel, and FIG. 271 shows the total diameter (tumor+leg) evaluation ofHT1080 tumors in mice treated with placebo, PM01183, dacarbazine, andthe corresponding combinations for the groups dosed at the two highestratios.

TABLE 21 % T/C on day Group Dose Test materials 0 2 4 7 9 11 14 16 G0110 ml/kg Placebo — — — — — — — — (Control group) G02 0.18 mg/kg PM01183100 59.3 40.0 26.9 26.7 11.5 21.2 30.6 G03 0.135 mg/kg PM01183 100 63.062.9 48.1 36.0 30.2 33.0 41.9 G04 0.09 mg/kg PM01183 100 66.7 57.1 65.448.0 42.7 45.8 56.4 G05 0.045 mg/kg PM01183 100 77.8 74.3 94.2 80.0 74.080.5 91.1 G06 150.0 mg/kg Dacarbazine 100 40.7 28.6 30.8 44.0 37.5 44.957.3 G07 112.5 mg/kg Dacarbazine 100 48.1 34.3 53.8 48.0 37.5 43.2 53.2G08 75.0 mg/kg Dacarbazine 100 74.1 65.7 69.2 58.7 45.8 46.6 51.6 G0937.5 mg/kg Dacarbazine 100 51.8 54.3 65.4 61.3 47.9 55.1 62.1 G10 0.18mg/kg PM01183 100 37.0 22.9 17.3 4.0 10.4 −4.2 1.0 150.0 mg/kgDacarbazine G11 0.135 mg/kg PM01183 100 29.6 25.7 11.5 −8.0 −17.7 −6.87.3 112.5 mg/kg Dacarbazine G12 0.09 mg/kg PM01183 100 37.0 31.4 28.852.0 43.7 50.8 64.5 75.0 mg/kg Dacarbazine G13 0.045 mg/kg PM01183 10055.6 51.4 67.3 70.7 62.5 59.3 62.1 37.5 mg/kg Dacarbazine Placebo: asdisclosed in table 13.

According to this assay it was found that:

a. The combination treatment of PM01183 and dacarbazine was effective inthe inhibition of the growth of the HT1080 fibrosarcoma cells, resultingin a statistically significant (P<0.01) reduction of total diameter(tumor+leg) compared to the control group with T/C values of 1.0% and7.3% (Day 16) in the two highly-dosed groups. Moreover, the combinationof PM01183 and dacarbazine produced lower T/C values than the moreactive single agent in this experiment (PM01183 at doses of 0.18 mg/kgand 0.135 mg/kg). Specifically, the TC (%) values of the combination(150 mg/kg dacarbazine+0.18 mg/kg PM01183) vs PM01183 alone (0.18 mg/kgPM01183) were 4.0 vs 26.7 (day 9), 10.4 vs 11.5 (day 11), −4.2 vs 21.2(day 14), and 1.0 vs 30.6 (day 16), and the TC (%) values of thecombination (112.5 mg/kg dacarbazine+0.135 mg/kg PM01183) vs PM01183alone (0.135 mg/kg PM01183) were −8.0 vs 36.0 (day 9), −17.7 vs 30.2(day 11), −6.8 vs 33.0 (day 14), and 7.3 vs 41.9 (day 16). Therefore,when PM01183 is combined with dacarbazine a potentiation of theantitumor activity is clearly observed.Additionally, based on the median-effect principle, the combination ofPM01183 and dacarbazine resulted in CI values of 0.28 (at Fa equal to0.97), indicating strong synergism in mice fibrosarcoma HT1080orthotopically implanted tumors.

Example 19. In Vivo Studies to Determine the Effect of PM01183 inCombination with Irinotecan in Human Colorectal Tumor Xenografts

The aim of these studies was to evaluate the ability of PM01183 topotentiate the antitumor activity of irinotecan by using a xenograftmodel of human colorectal carcinoma.Female athymic nude mice (Harlan Laboratories Models, S.L. (Barcelona,Spain) were utilized for all experiments. Animals were housed inindividually ventilated cages, up to ten per cage in a 12-hourlight-dark cycle at 21-23° C. and 40-60% humidity. The mice were allowedfree access to irradiated standard rodent diet and sterilized water.Animals were acclimated for at least 5 days prior to tumor implantationwith a tumor cell suspension.The tumor model used in these studies was HT-29 cell line, which wasobtained from the American Type Culture Collection (ATCC ref. HTB-38™).HT-29 cells were grown at 37° C. with 5% CO₂ in Dulbecco's modifiedEagle's medium (DMEM). Each animal was subcutaneously implanted on theright flank, using 26G needle and a 1 cc syringe, with 5×10⁶ HT-29cells, from in vitro passage 10, in 0.05 mL of 0.9% Sodium Chloride forinjection.Tumor measurements and treatment tolerability were performed anddetermined as disclosed in Example 13. Treatment tolerability wasassessed by monitoring body weight evolution, clinical signs as well asevidences of local damage in the injection site.When tumors reached a volume of about 180 mm³, mice were randomlyallocated into the treatments and control groups (N=5-7/group) based onbody weight and tumor volumen measurements by using NewLab OncologySoftware (version 2.25.06.00).PM01183 was provided in the form of vials of lyophilized PM01183 cakewhich was reconstituted with water for infusion to a concentration of0.2 mg/mL. The PM01183 stock solution was further diluted in 5% glucosesolution for injection to the dosing formulation concentrations.Irinotecan was provided in the form of a solution prepared by dilutingthe product with 5% glucose solution for injection to the target finalconcentration.In these experiments, PM01183 and irinotecan treatments, as well asplacebo, were intravenously administered as follows: PM01183 once perweek up to 3 consecutive weeks, on Days 0, 7 and 14, irinotecan wasdosed every 4 days, on Days 0, 4, 8, 12 and 16, and placebo wasadministered following the same schedule as those provided for PM01183and irinotecan. Dose level groups were administered either as singleagents or in combination.Comparison of the median tumor volume in the treatment groups (T) to themedian tumor volume in the control group (T/C×100%) was used forevaluation of the antitumor efficacy. In addition, potentiation wasdetermined as disclosed in Example 13.Table 22 reports the % T/C values obtained with PM01183 and irinotecanboth administered as single agents and in combination for each doselevel, and FIG. 272 shows the tumor volume evaluation of HT-29 tumors inmice treated with placebo, PM01183, irinotecan, and the correspondingcombinations for the groups dosed at the two highest ratios.

TABLE 22 Test % T/C on day Group Dose materials 0 3 5 7 10 G01 10 ml/kgPlacebo — — — — — (Control group) G02 0.18 mg/kg PM01183 100.4 108.486.5 101.1 116.5 G03 0.135 mg/kg PM01183 98.4 106.4 95.3 116.6 115.2 G040.09 mg/kg PM01183 98.4 100.9 88.7 121.9 129.8 G05 0.045 mg/kg PM0118399.8 103.7 100.6 111.1 135.8 G06 50.0 mg/kg Irinotecan 100.1 114.7 93.796.1 70.5 G07 37.5 mg/kg Irinotecan 98.4 108.1 97.5 99.2 84.3 G08 25.0mg/kg Irinotecan 98.8 108.6 97.2 101.4 96.5 G09 12.5 mg/kg Irinotecan99.0 99.1 90.6 97.4 92.7 G10 0.18 mg/kg PM01183 99.5 101.8 78.3 77.551.6 50.0 mg/kg Irinotecan G11 0.135 mg/kg PM01183 98.4 98.0 85.2 85.460.7 37.5 mg/kg Irinotecan G12 0.09 mg/kg PM01183 99.7 96.4 71.7 77.062.7 25.0 mg/kg Irinotecan G13 0.045 mg/kg PM01183 100.7 104.8 104.3116.0 98.8 12.5 mg/kg Irinotecan Test % T/C on day Group Dose materials12 14 17 20 G01 10 ml/kg Placebo — — — — (Control group) G02 0.18 mg/kgPM01183 115.4 123.4 86.7 77.5 G03 0.135 mg/kg PM01183 119.1 121.5 133.1105.2 G04 0.09 mg/kg PM01183 114.1 109.4 116.1 93.4 G05 0.045 mg/kgPM01183 125.1 109.3 G06 50.0 mg/kg Irinotecan 61.7 51.7 41.4 33.3 G0737.5 mg/kg Irinotecan 77.4 65.0 58.4 49.4 G08 25.0 mg/kg Irinotecan 79.382.5 76.3 60.3 G09 12.5 mg/kg Irinotecan 90.8 89.4 102.6 93.4 G10 0.18mg/kg PM01183 43.8 30.4 21.7 15.6 50.0 mg/kg Irinotecan G11 0.135 mg/kgPM01183 51.9 40.1 39.2 28.7 37.5 mg/kg Irinotecan G12 0.09 mg/kg PM0118357.7 50.1 47.2 40.7 25.0 mg/kg Irinotecan G13 0.045 mg/kg PM01183 85.590.5 88.1 76.5 12.5 mg/kg Irinotecan Placebo: as disclosed in table 13

According to this assay it was found that:

a. The combination treatment of PM01183 and irinotecan was effective inthe inhibition of the growth of the U87-MG brain tumor cells, resultingin a statistically significant (P<0.01) tumor reduction compared to thecontrol group with T/C values of 15.6% and 28.7% (Day 20) in the twohighly-dosed groups. Moreover, the combination of PM01183 and irinotecanproduced lower T/C values than the more active single agent in thisexperiment (irinotecan at doses of 50 mg/kg and 37.5 mg/kg).Specifically, the TC (%) values of the combination (50 mg/kgirinotecan+0.18 mg/kg PM01183) vs irinotecan alone (50 mg/kg irinotecan)were 30.4 vs 51.7 (day 14), 21.7 vs 41.4 (day 17), and 15.6 vs 33.3 (day20), and the TC (%) values of the combination (37.5 mg/kgirinotecan+0.135 mg/kg PM01183) vs irinotecan alone (37.5 mg/kgirinotecan) were 40.1 vs 65.0 (day 14), 39.2 vs 58.4 (day 17), and 28.7vs 49.4 (day 20). Therefore, when PM01183 is combined with irinotecan apotentiation of the antitumor activity is clearly observed.

Example 20. In Vitro Studies to Determine the Effect of PM01183 inCombination with Chemotherapeutic Agents on Human Leukemia Cell Lines

The following agents were evaluated in combination with PM01183:methotrexate, daunorubicin, aplidine, ET-743, PM02734 and PM00104 (stocksolutions of these compounds prepared in pure DMSO and stored at −20°C.). Additional serial dilutions were prepared in serum-free culturemedium to achieve a final 4× concentration. Aliquots of 50 μL of eachdiluted compound were added per well.

JURKAT and MOLT-4 were the human leukemia cell lines selected for thisassay, which were obtained from the American Type Culture Collection(ATCC). JURKAT and MOLT-4 cells were grown in phenol red-free RPMImedium supplemented with 10% Fetal Bovine Serum (FBS), 2 mM L-glutamineand 100 units/mL of Penicillin-Streptomycin, at 37° C., 5% CO2 and 95%humidity.The screening was performed in two parts:a. In the first set of assays, the relative potency of each compoundagainst the different cell lines was determined using a 72 hoursexposure in vitro cytotoxicity assay.Briefly, cells were seeded in 96 well microtiter plates at a density of50000 cells per well in 150 μL of culture medium and incubated for 4-6hours in drug-free medium before treatment with vehicle alone or testcompounds for 72 hours.After incubation, the cytotoxic effect was evaluated using a MTTreduction assay. 50 μL of MTT solution (1 mg/mL) were added to the wellsand incubated for 15-17 hours at 37° C. until formazan crystals wereformed. After gently removing the culture medium, DMSO was added todissolve the insoluble purple formazan product into a colored solution.The absorbance of the wells was quantified by measuring the opticaldensity at 540 nm. Results were expressed as percentage of control cellgrowth. The EC50 values (half-maximal effective concentration) used forthe combination studies were calculated using Prism v5.02 software(GraphPad). EC50 was expressed as molar concentration and representedthe mean of at least three independent assays.

The individual EC50 values obtained for each drug are shown in tables 23and 24.

TABLE 23 EC50 values in molar concentration (M) for each of the agentsfor the JURKAT tumor cell line. Com- Com- Compound EC50 (M) pound EC50(M) pound EC50 (M) Methotrexate 1.45E−07 Daunoru- 7.92E−07 Aplidine1.38E−08 bicin ET-743 6.96E−09 PM00104 4.83E−09 PM01183 1.55E−09 PM027345.50E−06

TABLE 24 EC50 values in molar concentration (M) for each of the agentsfor the MoLT-4 tumor cell line. Com- Com- Compound EC50 (M) pound EC50(M) pound EC50 (M) Methotrexate 4.39E−08 Aplidine 1.27E−09 ET-7433.84E−09 PM00104 1.55E−09 PM01183 8.57E−10 PM02734 1.44E−05b. In a second set of experiments, concentration-response curves for theagents tested, both alone and in two-drug combination, were performed,using the same methodology described in the previous paragraph.Given the significant differences between the respective EC50 values forPM01183 and the other standard drugs in this study, different ratios offixed concentrations for the two drugs were used. Normally, theselection of the fixed ratios of concentrations were the equipotentratio (1:1) at the EC50 value for each drug, and some other ratiosrepresenting different percentages of the corresponding EC50 values foreach drug above or below it. Using these starting concentrations,constant serial dilutions were performed to generate theconcentration-response curves for each set of drugs, alone and incombination.The effect of the two-drug combination, as compared with the effect ofeach drug alone, on the viability of tumor cells, was evaluated usingthe Chou and Talalay method which is based on the median-effectprinciple (Chou and Talalay, Adv. Enzyme Regul. 1984, 22, 27-55). Themedian-effect equation: f_(a)/f_(u)=(C/C_(m))^(m) (where C is the drugconcentration, C_(m) the median-effect concentration (i.e., IC50, ED50,or LD50, that inhibits the system under study by 50%), f_(a) the cellfraction affected by the drug concentration C, f_(u) the unaffectedfraction, and m the sigmoidicity coefficient of theconcentration-response curve), describes the relationship between theconcentration and the effect of a drug on a given biological system.Based on this equation, the term “combination index” (CI) is used as aquantitative measure of the degree of drug interactions. The combinationindex (CI) is determined by the equation:

CI=(C)₁/(C _(x))₁+(C)₂/(C _(x))₂

where (C_(x))₁ is the concentration of drug 1 alone that inhibits an xpercentage of a system, (C_(x))₂ the concentration of drug 2 alone thatinhibits the same x percentage of the system, and (C₁)+(C)₂ theconcentrations of drug 1 and drug 2 that in combination also inhibits anX percentage of the system. CI values were calculated by solving theequation for different values of f_(a) (i.e., for different degrees ofcell growth inhibition). CI values of <1 indicate synergy, the value of1 indicates additive effects, and values >1 indicate antagonism.Data were analyzed using CalcuSyn software (Biosoft, Cambridge, UK). Forstatistical analysis and graphs Prism software (GraphPad, San Diego,USA) was used. All the results represent the mean of at least threeindependent experiments.The effect of the tested drug combinations on cell proliferation isshown in FIGS. 273-283:Combination of PM01183 with Methotrexate.

The combination of PM01183 with methotrexate in JURKAT (FIG. 273) cellline resulted in some synergistic effects (CI<1) at determinedconcentrations of both drugs. The effects of PM01183 in combination withmethotrexate in MOLT-4 (FIG. 274) cell line were mostly additive.

Combination of PM01183 with Daunorubicin.

The combination of PM01183 with daunorubicin in JURKAT (FIG. 275) cellline was additive or synergistic (CI<1) at determined concentrations ofthe compounds.

Combination of PM01183 with Aplidine.

The combinations of PM01183 with aplidine in JURKAT (FIG. 276) andMOLT-4 (FIG. 277) cell lines resulted in some synergistic effects (CI<1)at determined concentrations of both drugs.

Combination of PM01183 with ET-743.

The combination of PM01183 with ET-743 in JURKAT (FIG. 278) cell linewas additive or synergistic (CI<1) at determined concentrations of bothdrugs. The combination of PM01183 with ET-743 in MOLT-4 (FIG. 279) cellline was mostly additive.

Combination of PM01183 with PM00104.

The combination of PM01183 with PM00104 in JURKAT (FIG. 280) cell linewas at least additive resulting in some synergistic effects (CI<1). Thecombination of PM01183 with PM00104 in MOLT-4 (FIG. 281) cell lineresulted in synergistic effects (CI<1).

Combination of PM01183 with PM02734.

The combination of PM01183 with PM02734 in JURKAT (FIG. 282) cell linewas mostly additive, resulting in some synergistic effects (CI<1) atdetermined concentrations of both drugs. The combination of PM01183 withET-743 in MOLT-4 (FIG. 283) cell line resulted in synergistic effects(CI<1).

Example 21. In Vitro Studies to Determine the Effect of PM01183 inCombination with Chemotherapeutic Agents on Human Lymphoma Cell Lines

The following agents were evaluated in combination with PM01183:gemcitabine, cytarabine, methotrexate, daunorubicin, ET-743, PM02734 andPM00104 (stock solutions of these compounds prepared in pure DMSO andstored at −20° C.). Additional serial dilutions were prepared inserum-free culture medium to achieve a final 4× concentration. Aliquotsof 50 μL of each diluted compound were added per well.RAMOS and U-937 were the human lymphoma cell lines selected for thisassay, which were obtained from the American Type Culture Collection(ATCC). RAMOS and U-937 cells were grown in phenol red-free RPMI mediumsupplemented with 10% Fetal Bovine Serum (FBS), 2 mM L-glutamine and 100units/mL of Penicillin-Streptomycin, at 37° C., 5% CO2 and 95% humidity.The screening was performed in two parts, as previously described inexample 20.

In the first set of assays, the individual EC50 values were determinedfor each drug as shown in tables 25 and 26.

TABLE 25 EC50 values in molar concentration (M) for each of the agentsfor the RAMOS tumor cell line. Com- Com- Compound EC50 (M) pound EC50(M) pound EC50 (M) Gemcitabine 2.51E−08 Cytarabine 3.64E−08 Metho-5.02E−06 trexate Daunoru- 3.15E−07 ET-743 9.55E−09 PM00104 4.35E−09bicin PM01183 1.39E−09 PM02734 1.36E−05

TABLE 26 EC50 values in molar concentration (M) for each of the agentsfor the U-937 tumor cell line. Com- Com- pound EC50 (M) Compound EC50(M) pound EC50 (M) Gemcita- 3.27E−08 Methotrexate 2.63E−08 Daunoru-3.04E−07 bine bicin ET-743 8.62E−09 PM00104 4.50E−09 PM01183 1.03E−09PM02734 6.85E−06

In the second set of assays, concentration-response curves for theagents tested, both alone and in two-drug combination, were performed.The effects of the drug combinations were evaluated using the Chou andTalalay method as described in the example 20

The effect of the tested drug combinations on cell proliferation isshown in FIGS. 284-296:Combination of PM01183 with Cytarabine.

The combination of PM01183 with cytarabine in RAMOS (FIG. 284) cell lineresulted in some synergistic effects (CI<1).

Combination of PM01183 with Methotrexate.

The combination of PM01183 with methotrexate in RAMOS (FIG. 285) cellline resulted in some synergistic effects (CI<1) at determinedconcentrations of both drugs. The effects of PM01183 in combination withmethotrexate in U-937 (FIG. 286) cell line resulted in some synergisticeffects at determined concentrations.

Combination of PM01183 with Gemcitabine.

The combination of PM01183 with gemcitabine in RAMOS (FIG. 287) cellline was additive or synergistic (CI<1) at determined concentrations ofboth drugs. The combination of PM01183 with gemcitabine in U-937 (FIG.288) cell line resulted in synergistic effects (CI<1).

Combination of PM01183 with Daunorubicin.

The combinations of PM01183 with daunorubicin in RAMOS (FIGS. 289) andU-937 (FIG. 290) cell lines were at least additive resulting in somesynergistic effects (CI<1).

Combination of PM01183 with ET-743.

The combinations of PM01183 with ET-743 in RAMOS (FIGS. 291) and U-937(FIG. 292) cell lines resulted in synergistic effects (CI<1) atdetermined concentrations of the compounds.

Combination of PM01183 with PM00104.

The combination of PM01183 with PM00104 in RAMOS (FIG. 293) resulted insynergistic effects (CI<1). The combination of PM01183 with PM00104 inU-937 (FIG. 294) cell line resulted in some synergistic effects (CI<1)at determined concentrations of both drugs.

Combination of PM01183 with PM02734.

The combination of PM01183 with PM02734 in RAMOS (FIG. 295) cell lineresulted in synergistic effects (CI<1), while the combination of PM01183with ET-743 in U-937 (FIG. 296) cell line was at least additive,resulting in some synergistic effects (CI<1) at high concentrations ofboth drugs.

What is claimed is:
 1. A method of treating cancer comprisingadministering to a patient in need of such treatment PM01183, or apharmaceutically acceptable salt thereof, and an anticancer antibiotic,wherein the amounts of PM01183 and the anticancer antibiotic whenadministered in combination are therapeutic.
 2. The method of treatingcancer according to claim 1, wherein the PM01183 and the cancerantibiotic are administered in synergistic amounts.
 3. The method oftreating cancer according to claim 1, wherein the cancer to be treatedis selected from the group consisting of lung cancer, sarcoma, malignantmelanoma, bladder carcinoma, prostate cancer, pancreas carcinoma,thyroid cancer, gastric carcinoma, ovarian cancer, hepatoma, breastcancer, colorectal cancer, kidney cancer, esophageal cancer,neuroblastoma, brain cancer, cervical cancer, anal cancer, testicularcancer, leukemia, multiple myeloma and lymphoma.
 4. The method oftreating cancer according to claim 3, wherein the cancer to be treatedis selected from the group consisting of lung cancer, sarcoma, malignantmelanoma, prostate cancer, pancreas carcinoma, gastric carcinoma,ovarian cancer, hepatoma, breast cancer, colorectal cancer, kidneycancer, brain cancer, leukemia and lymphoma.
 5. The method of treatingcancer according to claim 1, wherein PM01183, or a pharmaceuticallyacceptable salt thereof, and the anticancer antibiotic form part of thesame medicament.
 6. The method of treating cancer according to claim 1,wherein PM01183, or a pharmaceutically acceptable salt thereof, and theanticancer antibiotic are provided as separate medicaments foradministration at the same time or at different times.
 7. The method oftreating cancer according to claim 6, wherein PM01183, or apharmaceutically acceptable salt thereof, and the anticancer antibioticare provided as separate medicaments for administration at differenttimes.
 8. The method of treating cancer according to claim 1, whereinthe anticancer antibiotic is selected from the group consisting ofdaunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone,pixantrone, valrubicin, mitomycin C, bleomycin, actinomycin D andmithramycin.
 9. The method of treating cancer according to claim 8,wherein the anticancer antibiotic is selected from the group consistingof daunorubicin, doxorubicin, mitomycin C and actinomycin D.
 10. Themethod of treating cancer according to claim 9, wherein the anticancerantibiotic is doxorubicin.
 11. The method of treating cancer accordingto claim 10, wherein the combination of PM01183 and doxorubicin issynergistic.
 12. A method of increasing the therapeutic efficacy of ananticancer antibiotic in the treatment of cancer, which comprisesadministering to a patient in need thereof PM01183, or apharmaceutically acceptable salt thereof, in conjunction with saidanticancer antibiotic, wherein the amounts of the PM01183 and theanticancer antibiotic when administered in combination are therapeutic.13. The method of increasing the therapeutic efficacy of an anticancerantibiotic in the treatment of cancer according to claim 12, wherein thePM01183 and the anticancer antibiotic are administered in synergisticamounts.
 14. The method of increasing the therapeutic efficacy of ananticancer antibiotic in the treatment of cancer according to claim 12,wherein the cancer to be treated is selected from the group consistingof lung cancer, sarcoma, malignant melanoma, bladder carcinoma, prostatecancer, pancreas carcinoma, thyroid cancer, gastric carcinoma, ovariancancer, hepatoma, breast cancer, colorectal cancer, kidney cancer,esophageal cancer, neuroblastoma, brain cancer, cervical cancer, analcancer, testicular cancer, leukemia, multiple myeloma and lymphoma. 15.The method of increasing the therapeutic efficacy of an anticancerantibiotic in the treatment of cancer according to claim 14, wherein thecancer to be treated is selected from the group consisting of lungcancer, sarcoma, malignant melanoma, prostate cancer, pancreascarcinoma, gastric carcinoma, ovarian cancer, hepatoma, breast cancer,colorectal cancer, kidney cancer, brain cancer, leukemia and lymphoma.16. The method of increasing the therapeutic efficacy of an anticancerantibiotic in the treatment of cancer according to claim 12, whereinPM01183, or a pharmaceutically acceptable salt thereof, and theanticancer antibiotic form part of the same medicament.
 17. The methodof increasing the therapeutic efficacy of an anticancer antibiotic inthe treatment of cancer according to claim 12, wherein PM01183, or apharmaceutically acceptable salt thereof, and the anticancer antibioticare provided as separate medicaments for administration at the same timeor at different times.
 18. The method of increasing the therapeuticefficacy of an anticancer antibiotic in the treatment of canceraccording to claim 17, wherein PM01183, or a pharmaceutically acceptablesalt thereof, and the anticancer antibiotic are provided as separatemedicaments for administration at different times.
 19. The method ofincreasing the therapeutic efficacy of an anticancer antibiotic in thetreatment of cancer according to claim 15, wherein the anticancerantibiotic is selected from the group consisting of daunorubicin,doxorubicin, epirubicin, idarubicin, mitoxantrone, pixantrone,valrubicin, mitomycin C, bleomycin, actinomycin D and mithramycin. 20.The method of increasing the therapeutic efficacy of an anticancerantibiotic in the treatment of cancer according to claim 25, wherein theanticancer antibiotic is selected from the group consisting ofdaunorubicin, doxorubicin, mitomycin C and actinomycin D.
 21. The methodof increasing the therapeutic efficacy of an anticancer antibiotic inthe treatment of cancer according to claim 20, wherein the anticancerantibiotic is doxorubicin.
 22. The method of increasing the therapeuticefficacy of an anticancer antibiotic in the treatment of canceraccording to claim 21, wherein the combination of PM01183 anddoxorubicin is synergistic.
 23. A kit for use in the treatment of cancerwhich comprises a dosage form of PM01183, or a pharmaceuticallyacceptable salt thereof, a dosage form of an anticancer antibiotic, andinstructions for the use of both drugs in combination.
 24. The kitaccording to claim 23, wherein the anticancer antibiotic is selectedfrom the group consisting of daunorubicin, doxorubicin, epirubicin,idarubicin, mitoxantrone, pixantrone, valrubicin, mitomycin C,bleomycin, actinomycin D and mithramycin.
 25. The kit according to claim24, wherein the anticancer antibiotic is selected from the groupconsisting of daunorubicin, doxorubicin, mitomycin C and actinomycin D.26. The kit according to claim 25, wherein anticancer antibiotic isdoxorubicin.